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WO2002103054A1 - Marche sur le genome par l'amplification selective de bibliotheque d'adn de translation de coupure et l'amplification a partir de melanges complexes de matrices - Google Patents

Marche sur le genome par l'amplification selective de bibliotheque d'adn de translation de coupure et l'amplification a partir de melanges complexes de matrices Download PDF

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Publication number
WO2002103054A1
WO2002103054A1 PCT/US2001/044970 US0144970W WO02103054A1 WO 2002103054 A1 WO2002103054 A1 WO 2002103054A1 US 0144970 W US0144970 W US 0144970W WO 02103054 A1 WO02103054 A1 WO 02103054A1
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dna
sequence
nick
primer
nick translation
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PCT/US2001/044970
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English (en)
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Vladimir L. Makarov
Emmanuel Kamberov
Irina Sleptsova
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Rubicon Genomics Inc.
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Publication of WO2002103054A1 publication Critical patent/WO2002103054A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present invention relates generally to the fields of molecular biology and genomes. Particularly, it concerns utilization of DNA libraries for amplifying and analyzing DNA. More particularly, it concerns utilizing DNA libraries of nick translated products for chromosome walking.
  • the beneficial effect of amplifying mixtures of DNA is that it facilitates analysis of large pieces of DNA (e.g., chromosomes) by creating libraries of molecule that are small enough to be analyzed by existing techniques.
  • DNA e.g., chromosomes
  • the largest molecule that can be subjected to DNA sequencing methods is less than 2000 bases long, which is many orders of magnitude shorter than single chromosomes of organisms.
  • short molecules can be analyzed, considerable effort is required to assemble the information from the analysis of the short molecules into a description of the larger piece of DNA. 1.
  • Unique-sequence source DNA molecules can be amplified by separating them from other molecules (e.g., by electrophoresis), ligating them into an autonomously replicating genetic element (e.g., a bacterial plasmid), transfecting a host cell with the recombinant genetic element, and growing a clone of a single transfected host cell to product many copies of the genetic element having the insert with the same unique sequence as the source DNA (Sambrook, et al., 1989).
  • an autonomously replicating genetic element e.g., a bacterial plasmid
  • PCR polymerase chain reaction
  • DNA DNA sequence located between two known sequences
  • PCR involves the repetition of a cycle consisting of denaturation of the source (template) DNA, hybridization of two oligonucleotide primers to known sequences flanking the region to the amplified, primer extension using a DNA polymerase to synthesize strands complementary to the DNA region located between the two primer sites. Because the products of one cycle of amplification serve as source DNA for succeeding cycles, the amplification is exponential. PCR can synthesize large numbers of specific molecules quickly and inexpensively.
  • the major disadvantages of the PCR method to amplify DNA are that 1) information about two flanking sequences must be known in order to specify the sequences of the primers, 2) synthesis of primers is expensive, 3) the level of amplification achieved depends strongly on the primer sequences, source DNA sequence, and the molecular weight of the amplified DNA and 4) the length of amplified DNA is usually limited to less than 5 kb, although "long-distance" PCR (Cheng, 1994) allows molecules as long as 20 kb to be amplified.
  • One-sided PCR techniques are able to amplify unknown DNA adjacent to one known sequence. These techniques can be divided into 3 categories: a) ligation- mediated PCR, facilitated by addition of a universal adaptor sequence to a terminus usually created by digestion with a restriction endonuclease; b) universal primer-mediated PCR, facilitated by a primer extension reaction initiated at arbitrary sites c) terminal transferase- mediated PCR, facilitated by addition of a homonucleotide "tail" to the 3' end of DNA fragments; and d) "inverse PCR, facilitated by circularization of the template molecules. These techniques can be used to amplify successive regions along a large DNA template in a process sometimes called "chromosome walking.”
  • One-sided PCR can also be achieved by direct amplification using a combination of unique and non-unique primers.
  • Harrison et al. (1997) performed one-sided PCR using a degenerate oligonucleotide primer that was complementary to an unknown sequence and three nested primers complementary to a known sequence in order to sequence transgenes in mouse cells.
  • US5994058 specifies using a unique PCR primer and a second, partially degenerate PCR primer to achieve one-sided PCR.
  • Weber et al. (1998) used direct PCR of genomic DNA with nested primers from a known sequence and 1-4 primers complementary to frequent restriction sites. This technique does not require restriction digestion and ligation of adaptors to the ends of restriction fragments,
  • Terminal transferase can also be used in one-sided PCR.
  • Cormack and Somssich (1997) were able to amplify the termini of genomic DNA fragments using a method called RAGE (rapid amplification of genome ends) by a) restricting the genome with one or more restriction enzymes, b) denaturing the restricted DNA, c) providing a 3' polythymidine tail using terminal transferase, and d) performing two rounds of PCR using nested primers complementary to a known sequence as well as the adaptor.
  • RAGE rapid amplification of genome ends
  • RNA polymerase can also be used to achieve one-sided amplification of DNA.
  • U.S. Patent No. 6,027,913 shows how one-sided PCR can be combined with transcription with RNA polymerase to amplify and sequence regions of DNA with only one known sequence.
  • Inverse PCR is another method to amplify DNA based on knowledge of a single DNA sequence.
  • the template for inverse PCR is a circular molecule of DNA created by a complete restriction digestion, which contains a small region of known sequence as well as adjacent regions of unknown sequence.
  • the oligonucleotide primers are oriented such that during PCR they give rise to primer extension products that extend way from the known sequence. This "inside-out" PCR results in linear DNA products with known sequences at the termini.
  • Strand displacement amplification (Walker, et al. 1996a; Walker, et al. 1996b; U.S. Patent No. 5,648,213; U.S. Patent No. 6,124,120) is a method to amplify one of more termini of DNA fragments using an isothermal strand displacement reaction. The method is initiated at a nick near the terminus of a double-stranded DNA molecule, usually generated by a restriction enzyme, followed by a polymerization reaction by a DNA polymerase that is able to displace the strand complementary to the template strand. Linear amplification of the complementary strand is achieved by reusing the template multiple times by nicking each product strand as it is synthesized.
  • the products are strands with 5 ' ends at a unique site and 3' ends that are various distances from the 5' ends.
  • the extent of the strand displacement reaction is not controlled and therefore the lengths of the product strands are not uniform.
  • the polymerase used for strand displacement amplification does not. have a 5' exonuclease activity.
  • Rolling circle amplification (U.S. Patent No. 5,648,245) is a method to increase the effectiveness of the strand displacement reaction by using a circular template.
  • the polymerase which does not have a 5 ' exonclease activity, makes multiple copies of the information on the circular template as it makes multiple continuous cycles around the template.
  • the length of the product is very large—typically too large to be directly sequenced. Additional amplification is achieved if a second strand displacement primer is added to the reaction to used the first strand displacement product as a template.
  • Libraries are collections of small DNA molecules that represent all parts of a larger DNA molecule or collection of DNA molecules (Primrose, 1998; Cantor and Smith, 1999). Libraries can be used for analytical and preparative purposes.
  • Genomic clone libraries are the collection of bacterial clones containing fragments of genomic DNA.
  • cDNA clone libraries are collections of clones derived from the mRNA molecules in a tissue.
  • Cloning of non-specific DNA is commonly used to separate and amplify DNA for analysis.
  • DNA from an entire genome, one chromosome, a virus, or a bacterial plasmid is fragmented by a suitable method (e.g., hydrodynamic shearing or digestion with restriction enzymes), ligated into a special region of a bacterial plasmid or other cloning vector, transfected into competent cells, amplified as a part of a plasmid or chromosome during proliferation of the cells, and harvested from the cell culture.
  • a suitable method e.g., hydrodynamic shearing or digestion with restriction enzymes
  • This "shotgun" cloning method is used very frequently, because: 1) it is inexpensive, 2) it produces very pure sequences that are usually faithful copies of the source DNA, 3) it can be used in conjunction with clone screening techniques to create an unlimited amount of specific-sequence DNA, 4) it allows simultaneous amplification of many different sequences, 5) it can be used to amplify DNA as large as 1,000,000 bp long, and 6) the cloned DNA can be directly used for sequencing and other purposes. a. Multiplex cloning
  • Cloning is inexpensive, because many pieces of DNA can be simultaneously transfected into host cells.
  • the general term for this process of mixing a number of different entities is "multiplexing," and is a common strategy for increasing the number of signals or molecules that can be processed simultaneously and subsequently separated to recover the information about the individual signals or molecules.
  • the recovery process involves diluting the bacterial culture such that an aliquot contains a single bacterium carrying a single plasmid, allowing the bacterium to multiply to create many copies of the original plasmid, and isolating the cloned DNA for further analysis.
  • One clone from each transfection pool is combined with one clone from each of the other transfection pools in order to create a mixture of bacteria having a mixture of inserted sequences, where each specific inserted sequence is tagged with a unique vector sequence, and therefore can be identified by hybridization to the nucleic acid tag.
  • This mixture of cloned DNA molecules can be subsequently separated and subjected to any enzymatic, chemical, or physical processes for analysis such as treatment with polymerase or size separation by electrophoresis.
  • the information about individual molecules can be recovered by detection of the nucleic acid tag sequences by hybridization, PCR amplification, or DNA sequencing.
  • 5,714,318 is directed to a technique whereby the tag sequences are ligated to the DNA fragments before cloning using a universal vector.
  • PCT WO 98/15644 specifies a method whereby the tag sequences added before transfection are amplified using PCR after electrophoretic separation of the denatured DNA.
  • Disadvantages [0022] The disadvantage of preparing DNA by amplifying random fragments of DNA is that considerable effort is necessary to assemble the information within the short fragments into a description of the original, source DNA molecule.
  • Shotgun sequencing involves sequencing one or both ends of small DNA fragments that have been cloned from randomly-fragmented large pieces of DNA. During the sequencing of many such random fragments of DNA, overlapping sequences are identified from those clones that by chance contain redundant sequence information. As more and more fragments are sequenced more overlaps can be found from contiguous regions (contigs). As more and more fragments are sequenced the regions that are not represented become smaller and less frequent.
  • DNA libraries can be formed in vitro and subjected to various selection steps to recover information about specific sequences.
  • In vitro libraries are rarely used in genomics, because the methods that exist for creating such libraries do not offer advantages over cloned libraries.
  • the methods used to amplify the in vitro libraries are not able to amplify all of the DNA in an unbiased manner, because of the size and sequence dependence of amplification efficiency.
  • WO 00/18960 describes how different methods of DNA amplification can be used to create a library of DNA molecules representing a specific subset of the sequences within the genome for purposes of detecting genetic polymorphisms. "Random-prime PCR" (U.S. Patent No. 5,043,272; U.S. Patent No.
  • Single-molecule PCR can be used to amplify individual randomly- fragmented DNA molecules (Lukyanov et al, 1996).
  • the source DNA is first fragmented into molecules usually less than 10,000 bp in size, ligated to adaptor oligonucleotides, and extensively diluted and aliquoted into separate fractions such that the fractions often contain only a single molecule.
  • PCR amplification of a fraction containing a single molecule creates a very large number of molecules identical to one of the original fragments. If the molecules are randomly fragmented, the amplified fractions represent DNA from random positions within the source DNA.
  • WO 00/15779 A2 describes how a specific sequence can be amplified from a library of circular molecules with random genomic inserts using rolling circle amplification.
  • Directed cloning is a procedure to clone DNA from different parts of a larger piece of DNA, usually for the purpose of sequencing DNA from different positions along the source DNA.
  • Methods to clone DNA with "nested deletions" have been used to make "ordered libraries" of clones that have DNA starting at different regions along a long piece of source DNA.
  • one end of the source DNA is digested with one or more exonuclease activities to delete part of the sequence (McCombie et al., 1991; U.S. Patent No. 4,843,003). By controlling the extent of exonuclease digestion, the average amount of the deletion can be controlled.
  • the DNA molecules are subsequently separated based on size and cloned.
  • clomng molecules with different molecular weights, many copies of identical DNA plasmids are produced that have inserts ending at controlled positions within the source DNA.
  • Transposon insertion (Berg et al., 1994) is also used to clone different regions of source DNA by facilitating priming or cleavage at random positions in the plasmids.
  • the size separation and recloning steps make both of these methods labor intensive and slow. They are generally limited to covering regions less than 10 kb in size and cannot be used directly on genomic DNA but rather cloned DNA molecules.
  • the first method usually involves hybridization of one clone or other identifiable sequence to all other clones in a library. Those clones that hybridize contain overlapping sequences. This method is useful for locating clones that overlap a common site (e.g., a specific gene) in the genome, but is too laborious to create an ordered library of an entire genome. In addition many organisms have large amounts of repetitive DNA that can give false indications of overlap between two regions. The resolution of the hybridization techniques is only as good as the distance between known sequences of DNA.
  • the FISH method allows a particular sequence or limited set of sequences to be localized along a chromosome by hybridization of a fluorescently-labeled probe with a spread of intact chromosomes, followed by light-microscopic localization of the fluorescence. This technique is also only of use to locate a specific sequence or small number of sequences, rather than to create a physical map of the entire genome or an ordered library representing the entire genome.
  • the resolution of the light microscope limits the resolution of FISH to about 1,000,000 bp. To map a single-copy sequence, the FISH probe usually needs to be about 10,000 long.
  • Mapping by restriction digestion is frequently used to determine overlaps between clones, thereby allowing ordered libraries of clones to be constructed. It involves assembly of a number of large clones into a contiguous region (contig) by analyzing the overlaps in the restriction patterns of related clones. This method is insensitive to the presence of repetitive DNA.
  • the products of a complete or partial restriction digestion of every clone are size separated by electrophoresis and the molecular weights of the fragments analyzed by computer to find correlated sequences in different clones.
  • the information from the restriction patterns produced by five or more restriction enzymes is usually adequate to determine not only which clones overlap, but also the extent of overlap and whether some of the clones have deletions, additions, rearrangements, etc.
  • Sequence tagged sites are sequences, often from the 3' untranslated portions of mRNA, that can be uniquely amplified in the genome.
  • High-throughput methods employing sophisticated equipment have been devised to screen for the presence of tens of thousands of STSs in tens of thousands of clones. Two clones overlap to the extent that they share common STSs.
  • DNA sequencing is the most important analytical tool for understanding the genetic basis of living systems. The process involves determining the positions of each of the four major nucleotide bases, adenine (A), cytosine (C), guanine (G), and thymine (T) along the DNA molecule(s) of an organism. Short sequences of DNA are usually determined by creating a nested set of DNA fragments that begin at a unique site and terminate at a plurality of positions comprised of a specific base. The fragments te ⁇ ninated at each of the four natural nucleic acid bases (A, T, G and C) are then separated according to molecular size in order to determine the positions of each of the four bases relative to the unique site.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • the pattern of fragment lengths caused by strands that terminate at a specific base is called a "sequencing ladder.”
  • the interpretation of base positions as the result of one experiment on a DNA molecule is called a "read.”
  • the Maxim-Gilbert method involves degrading DNA at a specific base using chemical reagents. The DNA strands terminating at a particular base are denatured and electrophoresed to determine the positions of the particular base.
  • the Maxim-Gilbert method involves dangerous chemicals, and is time- and labor- intensive. It is no longer used for most applications.
  • the Sanger sequencing method is currently the most popular format for sequencing. It employs single-stranded DNA (ssDNA) created using special viruses like M13 or by denaturing double-stranded DNA (dsDNA).
  • ssDNA single-stranded DNA
  • dsDNA denaturing double-stranded DNA
  • An oligonucleotide sequencing primer is hybridized to a unique site of the ssDNA and a DNA polymerase is used to synthesize a new strand complementary to the original strand using all four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) and small amounts of one or more dideoxyribonucleotide triphosphates (ddATP, ddCTP, ddGTP, and/or ddTTP), which cause termination of synthesis.
  • dATP deoxyribonucleotide triphosphates
  • ddCTP deoxyribonucleotide tri
  • the DNA is denatured and electrophoresed into a "ladder" of bands representing the distance of the termination site from the 5 ' end of the primer. If only one ddNTP (e.g., ddGTP) is used only those molecules that end with guanine will be detected in the ladder. By using ddNTPs with four different labels all four ddNTPs can be incorporated in the same polymerization reaction and the molecules ending with each of the four bases can be separately detected after electrophoresis in order to read the base sequence.
  • ddNTP e.g., ddGTP
  • Sequencing DNA that is flanked by vector or PCR primer DNA of known sequence can undergo Sanger termination reactions initiated from one end using a primer complementary to those known sequences.
  • These sequencing primers are inexpensive, because the same primers can be used for DNA cloned into the same vector or PCR amplified using primers with common terminal sequences.
  • Commonly-used electrophoretic techniques for separating the dideoxyribonucleotide-terminated DNA molecules are limited to resolving sequencing ladders shorter than 500 - 1000 bases. Therefore only the first 500 - 1000 nucleic acid bases can be "read” by this or any other method of sequencing the DNA. Sequencing DNA beyond the first 500 - 1000 bases requires special techniques. 3.
  • Other base-specific termination methods are examples of bases that are flanked by vector or PCR primer DNA of known sequence.
  • sequence of a short fragment can be read by hybridizing different oligonucleotides with the unknown sequence, followed by deciphering the information to reconstruct the sequence.
  • This "sequencing by hybridization” is limited to fragments of DNA ⁇ 50 bp in length. It is difficult to amplify such short pieces of DNA for sequencing. However, even if sequencing many random 50 bp pieces were possible, assembling the short, sometimes overlapping sequences into the complete sequence of a large piece of DNA would be impossible.
  • the use of sequencing by hybridization is currently limited to resequencing, that is testing the sequence of regions that have already been sequenced.
  • Primer walking initiates the Sanger reaction at sequence-specific sites within long DNA.
  • most emphasis is on methods to amplify DNA in such a way that one of the ends originates from a specific position within the long DNA molecule.
  • a custom sequencing primer can be made that is complementary to the known part of the sequence, and used to prime a Sanger dideoxyribonucleotide termination reaction that extends further into the unknown region of the DNA.
  • This procedure is called "primer walking.”
  • the requirement to synthesize a new oligonucleotide every 400 - 1000 bp makes this method expensive. The method is slow, because each step is done in series rather than in parallel. In addition each new primer has a significant failure rate until optimum conditions are determined.
  • Primer walking is primarily used to fill gaps in the sequence that have not been read after shotgun sequencing or to complete the sequencing of small DNA fragments ⁇ 5,000 bp in length.
  • WO 00/60121 addresses using a single synthetic primer for PCR to genome walk to unknown sequences from a known sequence.
  • the 5 '-blocked primer anneals to the template and is extended, followed by coupling to the extended product of a 3 '- blocked oligonucleotide of known sequence, thereby creating a single stranded molecule having had only a single region of known target DNA sequence.
  • sequencing an amplified product from the extended product having the coupled 3 '-blocked oligonucleotide the process can be applied reiteratively to elucidate consecutive adjacent unknown sequences. 2.
  • PCR can be used to amplify a specific region within a large DNA molecule. Because the PCR primers must be complementary to the DNA flanking the specific region, this method is usually used only to prepare DNA to "resequence" a region of DNA.
  • clomng or PCR amplification of long DNA with nested deletions brought about by nuclease cleavage or transposon insertion enables ordered libraries of DNA to be created.
  • exonuclease is used to progressively digest one end of the DNA there is some control over the position of one end of the molecule.
  • the exonuclease activity cannot be controlled to give a narrow distribution in molecular weights, so typically the exonuclease-treated DNA is separated by electrophoresis to better select the position of the end of the DNA samples before cloning.
  • transposon insertion is nearly random, clones containing inserted elements have to be screened before choosing which clones have the insertion at a specific internal site.
  • the labor-intense steps of clone screening make these methods impractical except for DNA less than about 10 kb long.
  • the only practical method for preparing DNA longer than 5 kb for sequencing is subcloning the source DNA as random fragments small enough to be sequenced.
  • the large source DNA molecule is fragmented by sonication or hydrodynamic shearing, fractionated to select the optimum fragment size, and then subcloned into a bacterial plasmid or virus genome.
  • the individual subclones can be subjected to Sanger or other sequencing reactions in order to determine sequences within the source DNA. If many overlapping subclones are sequenced, the entire sequence for the large source DNA can be determined.
  • Genomes up to several millions or billions of base pairs in length can be randomly fragmented and subcloned as small fragments. However in the process of fragmentation all information about the relative positions of the fragment sequences in the native genome is lost. However this information can be recovered by sequencing with 5 - 10- fold redundancy (i.e., the number of bases sequenced in different reactions add up to 5 to 10 times as many bases in the genome) so as to generate sufficiently numerous overlaps between the sequences of different fragments that a computer program can assemble the sequences from the subclones into large contiguous sequences (contigs).
  • 5 - 10- fold redundancy i.e., the number of bases sequenced in different reactions add up to 5 to 10 times as many bases in the genome
  • the directed shotgun strategy adopted by the Human Genome Project, reduces the difficulty of sequence assembly by limiting the analysis to one large clone at a time.
  • This "clone-by-clone” approach requires four steps: 1) large-insert cloning, comprised of a) random fragmentation of the genome into segments 100,000 - 300,000 bp in size, b) cloning of the large segments, and c) isolation, selection and mapping of the clones; 2) random fragmentation and subcloning of each clone as thousands of short subclones; 3) sequencing random subclones and assembly of the overlapping sequences into contiguous regions; and 4) "finishing" the sequence by filling the gaps between contiguous regions and resolving inaccuracies.
  • the positions of the sequences of the large clones within the genome are determined by the mapping steps, and the positions of the sequences of the subclones are determined by redundant sequencing of the subclones and computer assembly of the sequences of individual large clones. Substantial initial investment of resources and time are required for the first two steps before sequencing begins. This inhibits sequencing DNA from different species or individuals. Sequencing random subclones is highly inefficient, because significant gaps exist until the subclones have been sequenced to about 7X redundancy. Finishing requires "smart" workers and effort equivalent to an additional ⁇ 3X sequencing redundancy.
  • the directed shotgun sequencing method is more likely to finish a large genome than is pure shotgun sequencing.
  • the computer effort for directed shotgun sequencing is more than 20 times less than that required for pure shotgun sequencing.
  • the DNA from an individual human or animal is amplified, usually by PCR, labeled with a detectable tag, and hybridized to spots of DNA with known sequences bound to a surface. If the individual's DNA contains sequences that are complementary to those on one or more spots on the DNA array, the tagged molecules are physically detected. If the individual's amplified DNA is not complementary to the probe DNA in a spot, the tagged molecules are not detected. Microarrays of different design have different sensitivities to the amount of tested DNA and the exact amount of sequence complementarity that is required for a positive result.
  • microarray resequencing technique is that many regions of an individual's DNA can be simultaneously amplified using multiplex PCR, and the mixture of amplified genetic elements hybridized simultaneously to a microarray having thousands of different probe spots, such that variations at many different sites can be simultaneously detected.
  • PCR single nucleotide polymorphism
  • SNP single nucleotide polymorphism
  • a second disadvantage to PCR is that only a limited number of DNA bases can be amplified from each element (usually ⁇ 2000 bp). Many applications require resequencing entire genes, which can be up to 200,000 bp in length.
  • nick translate molecule libraries More particularly, the present invention teaches generating a library of nick translate molecules to amplify and sequence for the purpose of obtaining successive overlapping sequences from a plurality of nick translate molecules.
  • the primary PENTAmer library in a specific embodiment, is prepared in vitro from bacterial or human genome using the teachings provided herein.
  • the primary PENTAmer library generated in vitro from a genome is amplified more than about 1000 times without any significant change in representation of the specific PENTAmer amplicons.
  • a primary PENTAmer library (directly or after amplification), such as from a bacteria or human, is used to amplify a specific PENTAmer or a PENTAmer sub-pool preferably using only one sequence-specific primer, which generates templates that reproducibly produce high quality sequencing data.
  • the methods described herein allow systematically generating from about 550 to 750 bases of a new sequence located downstream the primer.
  • a primary eukaryotic (human) PENTAmer library (directly or after amplification) is used to amplify a specific PENTAmer or a PENTAmer sub-pool using two (or more) nested sequence-specific primers.
  • a circularized eukaryotic (human) PENTAmer library is used to amplify a specific PENTAmer or a PENTAmer sub- pool using inverse PCR and two (or more) sequence-specific primers.
  • the present invention utilizes a library of nick translate molecules as a means to walk along a chromosome.
  • walk walking
  • chromosome walking or “genome walking” are directed to the generation of unknown sequence from a sample nucleic acid, such as a genome, in a sequential manner by starting from a known sequence, in specific embodiments termed herein as a "kernel,” sequencing by a first sequencing reaction (called a "read"), and generating a second sequencing read from a region of sequence obtained in the first read.
  • the two reads will overlap to some extent, and a consecutive series of such reactions results in the preferred walking embodiment of the invention.
  • the amplifiable nick translate molecule is generated by methods comprising at least fragmenting a DNA sample; attaching an adaptor to one end of the fragmented molecules, such as by covalent attachment, wherein the adaptor comprises a nick; nick translating with a DNA polymerase having 5' ⁇ 3' polymerase activity and 5' ⁇ 3' exonuclease activity; and attaching a second adaptor to the other end of the nick translated product.
  • the nick translate molecule may be amplified by primer sequences for the adaptors.
  • the nick is preferably generated by an adaptor comprising more than one oligonucleotide, wherein the oligonucleotide assembly has a nick between them, a skilled artisan recognizes that the nick may be generated by any standard means in the art.
  • nick translate molecule refers to nucleic acid molecules produced by coordinated 5' ⁇ 3' polymerase activity, such as DNA polymerase, and 5' ⁇ 3' exonuclease activity.
  • the two activities can be present within on enzyme molecule (such as DNA polymerase I or Taq DNA polymerase). In a preferred embodiment, they have adaptor sequences at their 5 ' and 3 ' termini.
  • nick translation refers to a coupled polymerization/degradation process that is characterized by a coordinated 5' ⁇ 3' DNA polymerase activity and a 5 ' ⁇ 3 ' exonuclease activity.
  • partial cleavage refers to the cleavage by an endonuclease of a controlled fraction of the available sites within a DNA template.
  • the extent of partial cleavage can be controlled by, for example, limiting the reaction time, the amount of enzyme, and/or reaction conditions.
  • a method of producing a consecutive overlapping series of nucleic acid sequences from a DNA sample comprising the steps of generating a first amplifiable nick translation product, wherein said nick translation of said first amplifiable nick translation product initiates from a known nucleic acid sequence in the DNA sample; determining at least a partial sequence from said first nick translation product; and generating at least a second amplifiable nick translation product, wherein said nick translation of said second amplifiable nick translation product initiates from the partial sequence of said first nick translation product.
  • a method of producing a library of consecutive overlapping series of nucleic acid sequences from a DNA sample comprising DNA molecules having a region comprising a known nucleic acid sequence comprising the steps of digesting DNA molecules of the DNA sample with a first sequence-specific endonuclease to generate a plurality of DNA fragments; generating a first amplifiable nick translation product, wherein said nick translation of said first amplifiable nick translation product initiates from the known nucleic acid sequence; determining at least a partial sequence from said first nick translation product; and generating one or more additional amplifiable nick translation products, wherein said nick translation of said one or more amplifiable nick translation products initiates from the partial sequence of a previous nick translation product.
  • the method further comprises the step of digesting DNA molecules with at least a second sequence-specific endonuclease, wherein the preceding overlapping nick translation product is generated from a DNA fragment from digestion with the first sequence-specific endonuclease or from digestion with the second sequence-specific endonuclease.
  • a method of producing a library of consecutive overlapping series of nucleic acid sequences comprising the steps of obtaining a DNA sample comprising DNA molecules having a region comprising a known nucleic acid sequence; partially cleaving the DNA molecules with a sequence- specific endonuclease to generate a plurality of DNA ends; separating the cleaved DNA molecules; generating a first amplifiable nick translation product, wherein said nick translation of said first amplifiable nick translation product initiates from a known nucleic acid sequence; determining at least a partial sequence from said first nick translation product; and generating one or more amplifiable nick translation products, wherein said nick translation of said one or more amplifiable nick translation products initiates from the partial sequence of a previous nick translation product.
  • the separation of the cleaved DNA molecules is according to size. In another specific embodiment, the size separation is by gel size fractionation. In an additional specific embodiment, the nick translation products are amplified. [0071] In another specific embodiment, the amplification of the nick translation product comprises polymerase chain reaction utilizing a first primer specific to a known sequence hi the nick translation product and a second primer specific to an adaptor sequence of the nick translation product. In an additional specific embodiment, at least one of the nick translation products is selectively amplified from the plurality of nick translation products. In a further specific embodiment, the nick translation product is single stranded.
  • the partial cleavage of the DNA molecules comprises cleaving for a selected time with a frequently cutting sequence-specific endonuclease, wherein the sequence-specificity of the endonuclease is to three or four nucleotide bases.
  • the partial cleavage of the DNA molecules comprises subjecting the DNA molecules to a methylase prior to subjection to a methylation-sensitive sequence-specific endonuclease.
  • the selective amplification comprises introducing to said plurality of nick translation products a plurality of primers, wherein the primers comprise nucleotide base sequence complementary to an adaptor sequence in the nick translation product; an additional variable 3' terminal nucleotide; and a label; hybridizing the primers to their complementary nucleic acid sequences in the adaptor to form a mixture of primer/nick translate molecule hybrids; and extending from a primer having the 3 ' terminal nucleotide complementary to the nucleotide in the nick translate molecule immediately adjacent to the adaptor sequence, wherein the hybridizing and extending steps form a mixture of unextended primer/nick translate molecule hybrids and extended primer molecule/nick translate molecule hybrids.
  • the method further comprises binding of the mixture by the label to a support; washing the support-bound mixture to remove the nick translate molecules; removing the support-bound extended molecule from the support.
  • the primer further comprises two or more variable 3' terminal nucleotides.
  • the method further comprises separating the nick translate molecules by size. In an additional specific embodiment, the size separation is by gel fractionation. In another specific embodiment, the method further comprises a step of subjecting the size-separated nick translate molecules to an additional amplification step. In a specific embodiment, the selective amplification step is by suppression PCR.
  • the suppression PCR utilizes a primer comprising a nucleic acid sequence for a primer specific for an adaptor sequence of the nick translate molecule; and nucleic acid sequence complementary to a region in a plurality of nick translate molecules, whereby the nucleic acid sequence is 5' to the sequence for a primer specific for an adaptor sequence of the nick translate molecule.
  • the at least one nick translate molecule is amplified by primer extension ligation reactions.
  • the method further comprises immobilization of the nick translation molecules onto a solid support.
  • the solid support is a magnetic bead.
  • the primer extension/ligation reactions comprise initiating and extending the primer extension reaction with a first primer which is complementary to sequence in a subset of the plurality of nick translate molecules, wherein the complementary sequence of the nick translate molecule is adjacent to a first adaptor end of the nick translate molecule; and ligating an oligonucleotide to the 5 ' end of the extension product, wherein the oligonucleotide comprises sequence complementary to the first adaptor of the nick translate molecule and also comprises a sequence for binding by a second primer, wherein the second primer binding sequence in the oligonucleotide is 5' to the first adaptor complementary sequence in the oligonucleotide.
  • the method further comprise amplifying the primer extended molecule.
  • the method further comprises separating the primer extended molecule from the plurality of nick translate molecule.
  • the nick translate molecules were generated in the presence of dU nucleotides, the primer extended molecule contains no dU nucleotides, and wherein the separating step comprises degradation of the plurality of nick translate molecules by dU-glycosylase.
  • the amplification step comprises polymerase chain reaction using the second primer and a primer complementary to a second adaptor of the nick translate molecule.
  • the ligation/primer extension reactions comprise ligating in a head-to-tail orientation a plurality of oligonucleotides to form an oligonucleotide assembly, wherein the oligonucleotides are complementary to nick translate molecule sequence adjacent to a first adaptor end of the nick translate molecule and wherein the nick translate molecule sequence is present in a subset of the plurality of nick translate molecules, wherein the nick translation molecule has the first adaptor on one terminal end and a second adaptor on the other terminal end; initiating and extending the primer extension reaction with the 3' end of the oligonucleotide assembly; and ligating an oligonucleotide to the 5' end of the extension product, wherein the oligonucleotide comprises sequence complementary to the first adaptor of the nick translate molecule and also comprises sequence for binding by a first primer, wherein the first primer binding sequence is 5 ' to the first adaptor complementary sequence in the oli
  • the method further comprises the steps of separating the primer extended molecule from the plurality of nick translate molecules; and amplifying the primer extended molecule.
  • the nick translate molecules were generated in the presence of dU nucleotides
  • the primer extended molecule contains no dU nucleotides
  • the separating step comprises degradation of the plurality of nick translate molecules by dU-glycosylase.
  • the amplification step comprises polymerase chain reaction using the first primer and a second primer complementary to the second adaptor of the nick translate molecule.
  • the primer extension/ligation reaction comprises initiating and extending the primer extension reaction with a first primer which is complementary to sequence in a subset of the plurality of nick translate molecules, wherein the nick translate molecule sequence is adjacent to a first adaptor end of the nick translate molecule; and ligating an oligonucleotide to the 5 ' end of the extension product, wherein the oligonucleotide comprises sequence complementary to the first adaptor of the nick translate molecule; sequence for binding by a second primer, wherein the second primer binding sequence is 5' to the sequence in (1); and a label at the 5' end.
  • the method further comprises the steps of separating the primer extended molecule from the plurality of nick translate molecules by the label of the oligonucleotide; and amplifying the primer extended molecule.
  • the label is biotin.
  • the separation further comprises streptavidin-coated magnetic beads.
  • the amplification step comprises polymerase chain reaction using the second primer and a third primer complementary to a second adaptor of the nick translate molecule.
  • a method of sequencing nucleic acid comprising the steps of obtaining a DNA sample comprising DNA molecules having a region comprising a known nucleic acid sequence; partially cleaving the DNA molecules with a sequence-specific endonuclease to generate a plurality of DNA ends; separating the cleaved DNA molecules; generating a first amplifiable nick translation product, wherein the first amplifiable nick translation product comprises an adaptor at each end, wherein the nick translation of said first amplifiable nick translation product initiates from a known nucleic acid sequence; determining at least a partial sequence from said first nick translation product; and generating one or more additional amplifiable nick translation products, wherein said nick translation of said one or more additional amplifiable nick translation products initiates from the partial sequence of a previous nick translation product; and sequencing the nick translation products, wherein the amplified nick translation product is not subjected to
  • the DNA sample is a genome. In another specific embodiment, there is a limited amount of DNA sample.
  • the amplification is by polymerase chain reaction, and one of the primers for the polymerase chain reaction is used as a primer for the sequencing reaction.
  • at least a portion of the adaptor sequence is removed from the amplified nick translation molecule. In another specific embodiment, the removal step comprises subjecting the amplified nick translation molecule to a 5 ' exonuclease.
  • a region of the adaptor sequence of the nick translate molecule comprises a dU nucleotide and the removal comprises degradation by dU-glycosylase.
  • a region of the adaptor sequence comprises a ribonucleotide and the removal comprises degradation by alkaline hydrolysis.
  • the region of the second adaptor sequence is in a 3 ' region of the second adaptor sequence.
  • a method of providing sequence for a gap in a genome sequence comprising the steps of obtaining a DNA sample of the genome comprising DNA molecules having a region comprising a known nucleic acid sequence adjacent to the gap; digesting the DNA molecules with a plurality of sequence-specific endonucleases to generate a plurality of DNA ends; generating a first amplifiable nick translation product, wherein said nick translation of said first amplifiable nick translation product initiates from the known nucleic acid sequence; determining at least a partial sequence from said first nick translation product; and generating one or more additional amplifiable nick translation products, wherein said nick translation of said one or more amplifiable nick translation products initiates from the partial sequence of a previous nick translation product, wherein at least one of the amplifiable nick translation products comprises sequence of the gap.
  • the genome is a bacterial genome. In a specific embodiment, the genome is a plant genome. In a specific embodiment, the genome is an animal genome. In a specific embodiment, the animal genome is a human genome. In an additional specific embodiment, the bacteria are unculturable. In an additional specific embodiment, the bacteria is present in a plurality of bacteria.
  • a method of producing a library of consecutive overlapping series of nucleic acid sequences from a DNA sample comprising the steps of obtaining the DNA sample comprising a DNA molecule; digesting the DNA molecule with a first sequence-specific endonuclease to generate a plurality of DNA fragments, wherein at least one DNA fragment has a region comprising a known nucleic acid sequence; attaching a first adaptor molecule to ends of the DNA fragments to provide a nick translation initiation site, wherein the first adaptor comprises a label; subjecting the first adaptor-bound DNA fragment to nick translation comprising DNA polymerization and 5'-3' exonuclease activity, wherein the nick translation initiates from the known nucleic acid sequence, to generate a first nick translation product; isolating the nick translation product by the label; attaching a second adaptor molecule to the first nick translate product; determining at least a partial sequence from the first nick translation
  • a method of producing a library of consecutive overlapping series of nucleic acid sequences comprising the steps of obtaining a DNA sample comprising DNA molecules having a region comprising a known nucleic acid sequence; partially cleaving the DNA molecules with a sequence-specific endonuclease to generate a plurality of DNA fragments, wherein at least one DNA fragment has a region comprising a known nucleic acid sequence; separating the cleaved DNA fragments; attaching a first adaptor molecule to ends of the DNA fragments to provide a nick translation initiation site, wherein the first adaptor comprises a label; subjecting the first adaptor-bound DNA fragment to nick translation comprising DNA polymerization and 5'-3' exonuclease activity, wherein the nick translation initiates from the known nucleic acid sequence, to generate a first nick translation product; isolating the nick translation product by the label; attaching a second adaptor molecule to the first
  • the separation of the DNA fragments is by size. In another specific embodiment, the size separation is by electrophoresis. [0083] In another object of the present invention, there is a library of consecutive overlapping series of nucleic acid sequences from a DNA sample, wherein the library is generated by the methods described herein.
  • FIG. 1 illustrates genome walking by sequential amplification of the overlapping PENTAmers.
  • FIG. 2 demonstrates types of PENTAmer libraries.
  • FIGS. 3 A and 3B illustrate the general strategy of genome walking by a targeted amplification of the overlapping PENTAmers.
  • FIGS. 4A and 4B illustrate synthesis of the primary PENTAmer library from a genomic DNA completely digested with a restriction endonuclease.
  • FIGS. 5 A and 5B show synthesis of the primary PENTAmer library from a partially digested genomic DNA.
  • FIG. 6 demonstrates premature termination of the PENTAmer synthesis on short DNA fragments.
  • FIG. 7 illustrates amplification of the PENTAmer library produced by a partial restriction digestion using conventional PCR.
  • FIGS. 8 A and 8B show one-base selection by primer-extension/affinity capture procedure.
  • FIG. 9 demonstrates reducing the PENTAmer library complexity by primer extension polymerase chain reaction with primer-selector A.
  • FIG. 10 illustrates genome walking using overlapping PENTAmer library, conventional PCR, and DNA size fractionation-pooling strategy.
  • FIG. 11 illustrates amplification of the PENTAmer library produced by a partial restriction digestion using suppression PCR.
  • FIG. 12 illustrates preparation of the immobilized single-strand complementary PENTAmer library for the selection-amplification procedure.
  • FIGS. 13 A and 13B shows targeted PENTAmer amplification by primer extension-ligation-Method I.
  • FIGS. 14A and 14B demonstrates targeted PENTAmer amplification by modular oligonucleotide assembly-Method II.
  • FIGS. 15A and 15B demonstrates targeted PENTAmer amplification by modular oligonucleotide assembly-Method III.
  • FIGS. 16A and 16B demonstrates PENTAmer selection by primer extension/ligation followed by magnetic bead capture.
  • FIG. 17 shows sequencing of two overlapping fragments L and S generated by amplification of PENTAmer library (following partial restriction digestion) using unique primer P and universal primer B.
  • FIG. 18 illustrates sequencing gaps in a genome, such as a bacterial genome, using primary PENTAmer libraries.
  • FIG. 19 demonstrates positional genome walking by targeted PENTAmer amplification.
  • FIG. 20 demonstrates PCR amplification of genomic BarnR I PENTAmer E. coli library and selected kernel sequences.
  • FIG. 21 illustrates schematic presentation of assembly of short oligonucleotides on E. coli BamB. I PENTAmer library template.
  • FIG. 22 demonstrates assembly of short oligonucleotides at specific E. coli genomic kernel sequence by thermo-stable DNA ligase using secondary E. coli genomic Ban ⁇ I PENTamer library as template.
  • FIG. 23 shows selection of specific E. coli PENTAmer sequence by assembly of short oligonucleotides followed by extension with DNA polymerase and ligation of universal adaptor oligonucleotide at adaptor A using secondary E. coli genomic BamH I PENTAmer library as template.
  • FIG. 24 demonstrates PCR analysis of forty kernel sites in primary PENTAmer library from E. coli Sau3A I partial genomic digest.
  • FIG. 25 shows PCR analysis of two kernel sites in PENTAmer library from E. coli Sau3A I partial genomic digest after size separation.
  • FIG. 26 demonstrates PCR analysis of three kernel sequences selected by multiplexed linear amplification from secondary E. coli PENTAmer library derived from Saui I partial digest.
  • FIG. 27 shows PCR amplification of PENTAmer libraries prepared from human genomic DNA after partial Sau3A I or complete Ban ⁇ R I restriction digest.
  • FIG. 28 shows circularization of single-stranded human genomic DNA Sau3A I PENTAmer library.
  • FIG. 29 demonstrates PCR amplification of single-stranded circular Sau3A I human PENTAmer library and a kernel sequence.
  • FIG. 30 shows nested PCR amplification of kernel human genomic sequence from primary BamH. I and Sau3A I PENTAmer libraries.
  • FIG. 31 illustrates schematic presentation of regions in the 10 Kb human tp53 gene amplified by nested PCR from primary BamB. I and Sau3A I libraries.
  • a or “an” may mean one or more.
  • the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
  • another may mean at least a second or more.
  • nick translate molecule is used interchangeably with the terms “PENTAmer” or “nick translate product.”
  • the present invention is directed to chromosome walking through the generation of nick translate molecules, and a skilled artisan recognizes that the nick translate molecules may be generated by any standard means in the art. However, in a preferred embodiment, the nick translate molecules are adaptor attached nick translate molecules (designated a PENTAmer).
  • a primary PENTAmer is generated by:
  • the PENT reaction is initiated, continued, and terminated on a largely double-stranded template, which gives the PENTAmer amplification important advantages for creating DNA for sequence analysis.
  • An advantage of using PENTAmers to amplify different regions of the template is the fact that in most applications PENTAmers having different internal sequences have the same terminal sequences. These advantages are important for creating PENTAmers that are most useful as intermediates for in vitro or in vivo amplification. Amplification of these intermediates is more useful than direct amplification of DNA by cloning or PCR.
  • the PENTAmers can be degraded by incorporating distinguishable nucleotides during the reaction. For example, incorporation of dU nucleotides and subsequent exposure to dU-glycosylase allows destruction of the PENTAmers for separation from, for example, a desired nucleic molecule lacking the dU nucleotides.
  • the initiation site for a PENT reaction can be introduced by any method that results in a free 3' OH group on one side of a nick or gap in otherwise double-stranded DNA, including, but not limited to such groups introduced by: a) digestion by a restriction enzyme under conditions that only one strand of the double-stranded DNA template is hydrolyzed; b) random nicking by a chemical agent or an endonuclease such as DNAase I; c) nicking by fi gene product II or homologous enzymes from other filamentous bacteriophage (Meyer and Geider, 1979); and/or d) chemical nicking of the template directed by triple-helix formation (Grant and Dervan, 1996).
  • PENTAmer synthesis the primary means of initiation is through the ligation of an oligonucleotide primer onto the target nucleic acid.
  • This very powerful and general method to introduce an initiation site for strand replacement synthesis employs a panel of special double-stranded oligonucleotide adaptors designed specifically to be ligated to the termini produced by restriction enzymes. Each of these adaptors is designed such that the 3' end of the restriction fragment to be sequenced can be covalently joined (ligated) to the adaptor, but the 5' end cannot.
  • the 3' end of the adaptor remains as a free 3' OH at a 1 nucleotide gap in the DNA, which can serve as an initiation site for the strand-replacement sequencing of the restriction fragment.
  • a set of such adaptors for strand replacement initiation can be synthesized with labels (radioactive, fluorescent, or chemical) and incorporated into the dideoxyribonucleotide-terminated strands to facilitate the detection of the bands on sequencing gels.
  • adaptors with 5' and 3' extensions can be used in combination with restriction enzymes generating 2-base, 3 -base and 4-base (or more) overhangs.
  • the sense strand of the adaptor has a 5' phosphate group that can be efficiently ligated to the restriction fragment to be sequenced.
  • the anti-sense strand (bottom, underlined) is not phosphorylated at the 5' end and is missing one base at the 3' end, effectively preventing ligation between adaptors. This gap does not interfere with the covalent joining of the sense strand to the restriction fragment, and leaves a free 3' OH site in the anti-sense strand for initiation of strand replacement synthesis.
  • Polymerization may be terminated specific distances from the priming site by inhibiting the polymerase a specific time after initiation.
  • Taq DNA polymerase is capable of strand replacement at the rate of 250 bases/min, so that arrest of the polymerase after 10 min occurs about 2500 bases from the initiation site. This strategy allows for pieces of DNA to be isolated from different locations in the genome.
  • PENT reactions may also be terminated by incorporation of a dideoxyribonucleotide instead of the homologous naturally-occurring nucleotide. This terminates growth of the new DNA strand at one of the positions that was formerly occupied by dA, dT, dG, or dC by incorporating ddA, ddT, ddG, or ddC.
  • the reaction can be terminated using any suitable nucleotide analogs that prevent continuation of DNA synthesis at that site.
  • Secondary PENTAmers are created by two nick-translation reactions.
  • the length of the first PENT reaction determines the distance of one end of the secondary PENTAmer from the initiation position, whereas the second (shorter) PENT reaction determines the length of the secondary PENTAmer.
  • the advantage of secondary PENTAmers is that the position of the PENTAmer within the template DNA and the length of the PENTAmer are independently controlled.
  • a secondary PENTAmer is created and amplified by:
  • a secondary PENTAmer is created by:
  • the difficulty of immobilizing very large DNA fragments may be overcome by bringing together sequences from both the proximal and distal ends of long templates to create a recombinant PENTAmer.
  • a recombinant PENTAmer is made on a single template molecule, having different structures at the left (proximal) and right (distal) ends.
  • the initiation domain of adaptor RA is used to synthesize a PENTAmer containing the distal template sequences.
  • PENTAmers will only be created on those fragments that have been ligated to both ends of the recombination adaptor RA. Specific designs and use of recombination adaptors would be apparent to a skilled artisan.
  • One embodiment uses an adaptor RA comprising a first ligation domain complementary to the proximal tenninus of the template, an activatable second ligation domain complementary to the distal terminus, and a nick-translation initiation domain capable of translating the nick from the distal end toward the center of the template.
  • the template would be made resistant to cleavage by the activation restriction enzyme by methylation at the restriction recognition sites, and the second step would be executed in the following way: 1) removal of unligated adaptor RA from solution, 2) activation of adaptor RA by restriction digestion of the unmefhylated site within the adaptor, 3) dilution of the template, 4) ligation of the second ligation domain to the distal end of the template, and 5) concentration of the circularized molecules.
  • Step 3 is executed by the same methods used to create a primary PENTAmer, however the nick-translation initiates at the initiation domain of an RA adaptor.
  • the PENTAmer formed can be amplified by any of the methods described earlier, e.g., by PCR using primers complementary to sequences in adaptors.
  • a preferred design of a nick-translation adaptor is formed by annealing 3 oligonucleotides (or more): oligonucleotide 1, oligonucleotide 2 and oligonucleotide 3.
  • Oligonucleotide 1 has a phosphate group (P) at the 5' end and a blocking nucleotide at the 3' end, a non-specified nucleotide composition and length from about 10 to 200 bases.
  • Oligonucleotide 2 has a blocked 3' end, a non-phosphorylated 5' end, a nucleotide sequence complementary to the 5' part of oligonucleotide 1 and length from about 5 to 195 bases.
  • oligonucleotides 1 and 2 form a double-stranded end designed to be ligated to the 3' strand at the end of a template molecule.
  • a nick-translation adaptor can have blunt, 5'-protruding or 3'- protruding end.
  • Oligonucleotide 3 has a 3' hydroxyl group, a non-phosphorylated 5' end, a nucleotide sequence complementary to the 3' part of oligonucleotide 1, and length from about 5 to 195 bases.
  • Oligonucleotides 2 and 3 form a nick or a few base gap within the lower strand of the adaptor.
  • Oligonucleotide 3 can serve as a primer for initiation of the nick-translation reaction.
  • nick-attaching adaptors are partially double-stranded or completely single-stranded short DNA molecules that can be covalently linked to the 3' hydroxyl group of the nick-translation DNA product.
  • nick-translation DNA product can be a single-stranded molecule isolated from its DNA template or the nick-translation product still hybridized to the template DNA.
  • the nick-attaching adaptors are designed to complete the synthesis of the 3' end of PENTAmers.
  • PENTAmer walking is achieved by priming-selection and amplification of a limited number of PENTAmer molecules with a known sequence at their 5' end (FIG. 1). At every step a new DNA sequence located downstream from the primer(s) is generated. In a preferred embodiment, the predicted size of the amplicon guarantees the success of each walking step; that is, the amount of sequence information generated at each step is equal to the PENTAmer amplicon size (for example, 1 kb). In practice, the new sequence identified at each walking step is limited by existing DNA sequencing technology and usually does not exceed about 750 bp.
  • the nick- translate library should be redundant to the extent that at each step the 5 ' end of the nick- translate molecule can be identified, the molecule primed, amplified and sequenced. In principle, one library and one amplification is necessary at each step.
  • the corresponding primary PENTAmer library would result in a different level of coverage of genomic DNA.
  • the PENTAmer library prepared from DNA fragments after Sfi I and BamB. I digestion will have an average of about two PENTAmer molecules per 60 kb and 10 kb, respectively (FIG. 2 A and 2B) leaving substantial gaps between consecutive PENTAmer molecules (PENTAmers generated at both strands of DNA are herein considered separately: C- and W-PENTAmers).
  • the PENTAmer library prepared after partial restriction digestion of DNA with a frequently cutting endonuclease Sau3A I will have an average 8 molecules per 1 Kb. At the size of the PENTAmer amplicon of 1 Kb, the levels of redundancy for those cases A, B and C shown on FIG. 2 are 0.03, 0.2 and 8, respectively.
  • FIGS. 4A and 4B illustrate the preparation of the primary PENTAmer library for a given restriction enzyme R n presented in the following Protocol 1 :
  • Protocol 1 Preparation of the primary PENTAmer libraries by a complete digestion with different restriction enzymes c. Split DNA into N tubes containing N different restriction enzymes and corresponding buffer, and digest to completion.
  • the most suitable enzymes are the restriction endonucleases with 6-base specificity as, for example, BamB I, EcoR I, Hind III, etc. A skilled artisan is aware that there are more than 100 enzymes of this type currently available on the market. Stop the reaction by adding EDTA or/and by heating at 65-75° C.
  • d Incubate DNA samples with the alkaline phosphatase for an appropriate time to remove the phosphate group from all 5 ' DNA restriction fragments (this step is optional). Purify.
  • DNA by phenol/chlorophorm extraction-ethanol precipitation or using commercially available DNA purification kits.
  • e. Ligate the nick-translation adaptor A to all DNA ends. Purify DNA.
  • f. Incubate with a DNA polymerase possessing 5' exonuclease activity (for example, non-mutated Taq DNA polymerase) for a specific time to synthesize DNA molecules of a controlled size (PENT products).
  • PENT products for example, non-mutated Taq DNA polymerase
  • h. Ligate the second adaptor B to the 3' ends of immobilized PENT molecules.
  • N different primary PENTAmer sub-libraries are generated.
  • the sub-libraries can be additionally amplified if necessary using universal primers A and B.
  • FIG. 3 A illustrates the case when 10 individual PENTAmer libraries constitute a walking nick-translate DNA library.
  • the figure shows a DNA region covered by 21 PENTAmer amplicons originated from the bottom C-strand of DNA.
  • the walking process starts at the right end where the DNA sequence is known.
  • the selection of the specific PENTAmer molecule P n is achieved in the two steps: first, when choosing the corresponding sub-library R n for the amplification; and second, when amplifying the DNA fragment using sequence-specific primer Pr(n) and universal adaptor-specific primer B. Because there is no overlap between PENTAmers within one sub-library the exact location of the sequence- specific primer is not important except that it should anneal to DNA downstream the restriction site.
  • amplification and sequencing of the molecule V using sub- library R ⁇ and primers Pr 1 and B is resulted in identification of the restriction site j within the 3 ' end of the same molecule.
  • individual sub-library R and primers Pr 2 and B are used to amplify and sequence the molecule P .
  • the restriction site R 6 is identified at the 3 ' end of the P 4 DNA molecule and the P 6 molecule is amplified and sequenced using library R 6 and primers Pr 3 and B.
  • a redundant nick-translate DNA library is prepared by a partial digestion of DNA with one frequently cutting restriction endonuclease R (FIG. 3B).
  • the drawing shows 21 nick-translate molecules originated from the bottom C-DNA strand.
  • FIGS. 5 A and 5B illustrate the preparation of primary PENTAmer library produced by a partial digestion of DNA with a restriction enzyme R presented in the Protocol 2:
  • Protocol 2 Preparation of the primary PENTAmer library by a partial digestion with a frequently cutting restriction enzyme
  • a. Digest DNA partially with a frequently cutting restriction enzyme with 4 or 3 base specificity using limited time or limited enzyme strategy, or using a combined restriction digestion / methylation method.
  • suitable enzymes such as Sau3A I, Nla III, Cvi J, etc. Stop the reaction.
  • b. Incubate DNA samples with the alkaline phosphatase for an appropriate time to remove the phosphate group from all 5 ' DNA restriction fragments (this step is optional). Purify DNA by phenol/chloroform extraction-ethanol precipitation or using commercially available DNA purification kits.
  • nick-translation adaptor A Ligate the nick-translation adaptor A to all DNA ends. Purify DNA. d. Fractionate DNA by a gel electrophoresis to isolate fragments larger than double size of a PENTAmer molecules. The PENTAmers from smaller restriction fragments will be shorter than the expected PENTAmer size because of a premature collapse of two nick-translation reactions initiated at the opposite ends of the DNA fragments. e. Incubate with a DNA polymerase possessing 5' exonuclease activity (for example, non-mutated Taq DNA polymerase) for a specific time to synthesize DNA molecules of a controlled size (PENT products). f. Isolate PENT molecules by capturing on the streptavidin- coated magnetic beads. g. Ligate the second adaptor B to the 3' ends of immobilized PENT molecules. Wash.
  • the PENTAmers prepared from a partially digested DNA are substantially overlapped and form a highly redundant DNA library.
  • the size fractionation step is important because partial digestion generates DNA molecules of all sizes with about the same probability.
  • the PENTAmers from DNA fragments with the size smaller than double size of the expected PENTAmer amplicon length will be shorter because of a premature collapse of two nick-translation reactions initiated at the opposite ends of the DNA fragments (FIGS. 6B and 6C).
  • the overlapping PENTAmer library is used to walk along a chromosome.
  • the walking strategy would be very similar to that described in a previous section if there is a way to selectively amplify individual PENTAmer molecules.
  • FIG. 3B shows 21 overlapping PENTAmer molecules from the library generated by partial digestion of DNA with a restriction endonuclease R (only PENTAmers from the bottom strands are illustrated).
  • a minimal tiling path in this case can be created by a selective amplification and sequencing of the molecules P ls P 5 , P 9 , P 13 , P 17 and P 21 from a single nick- translate library R.
  • the present invention is also directed to solving the problem of sequencing complex mixtures of PENTAmers which are easy to generate by a conventional PCR.
  • Amplification of overlapping PENTAmers by standard PCR using one sequence-specific and one universal primer would result in selection and amplification of several molecules, specifically, a nested set of DNA fragments of different length which share the same priming site P (FIG. 7).
  • a nested set of DNA fragments of different length which share the same priming site P (FIG. 7).
  • FIG. 7 For example, from eight overlapping PENTAmer molecules shown on FIG. 7 only the molecules ## 2 to 7 will serve as templates for a primer- extension reaction with primer P. It is not obvious that the amplified molecules ## 2 - 7 (FIG. 7) could be directly used for DNA sequencing using primer P (or nested primer P') as a sequencing primer. Two factors could potentially affect the quality and length of the resulting sequencing ladder.
  • the bias towards a preferential amplification of the shortest DNA fragments could reduce the length of DNA sequencing.
  • the method relies on the segregation of PENTAmer molecules into sub- fractions according to a base composition at the region adjacent to the restriction site.
  • the segregation is achieved by selective priming and synthesis of DNA molecules using a set of biotinylated selective primers A* and universal primer B.
  • selective primers are complementary to the adaptor sequence A and the restriction site plus have an extra selective base(es) at their 3 ' end.
  • four one-base selective primers shown on FIGS. 8 A and 8B have in addition an extra G, A, T or C base at the 3' end.
  • Sixteen two- base selective primers have two additional selective bases at the 3 ' end, and so on.
  • the first step involves hybridization and extension of primer-selectors using wild type Taq DNA polymerase (FIGS. 8 A and 8B). The reactions proceed in four different tubes.
  • the next level of selection can be achieved by cleaving off the biotin moiety, releasing selected molecules into solution and repeating the selection step with a new set of selective primers. For example, after segregation of the PENTAmer library into 4 pools “G”, “A”, “T”, and “C” using one-base selective primers, the sub-libraries can be further segregated into 16 pools using two-base selective primers (FIG. 9).
  • the molecules for size fractionation can be generated also by n primer- extension reactions with sequence-specific primers Pi, P 2 , ..., P n or even one multiplexed polymerase-extension reaction using primers Pi, P 2 , ..., P n combined together in a one tube.
  • An additional approach to reduce the representation of short DNA fragments is to use a suppression PCR (Siebert et al., 1995) wherein the sequence-specific primer PS is designed to have an additional 5 ' sequence which is identical to the sequence of the universal adaptor primer B (FIG. 11).
  • the reaction is initiated by limited number of linear amplifications using sequence-specific suppression-PCR primer PS (FIG. 11) and completed by using suppression PCR mode with the universal primer B (FIG. 11). Because of formation of a specific panhandle DNA structure at the ends of DNA fragments the amplification of the shortest DNA fragments is suppressed and only large DNA molecules would be amplified (FIG. 11).
  • Suppression PCR offers an additional level of selection, namely, selection according to DNA fragment size.
  • FIGS. 13 A and 13B show the first targeted amplification method. It involves four major steps.
  • Step 1 Polymerase extension reaction with phosphorylated primer- selector P x complementary to the left side of the restriction site R x (FIG. 13A and 13B). Priming occurs internally within several overlapping PENTAmer molecules except PENTAmer X where priming occurs at the "restriction" end of the DNA fragment in the region immediately adjacent to the adaptor sequence A.
  • Step 2 Ligation of the tagged oligonucleotide P A to the 5 ' end of the extension product.
  • Oligonucleotide P A is complementary to the adaptor A, and it is ligated only to the terminally extended molecule on the targeted PENTAmer X (FIG. 13C).
  • Step 3 Degradation of the template PENTAmer DNA library by incubation with dU-glycosylase, followed by heating (FIG. 13D)
  • Step 4 PCR amplification using primers B and C (5' portion of the tagged oligo P A ) (FIG. 13E). b. Method 2
  • FIGS. 14A through 14E illustrate second protocol for the targeted amplification of PENTAmers. It has five major steps.
  • Step 1 Ligation-assembly reaction using short phosphorylated oligonucleotides Pi, P 2 , P 3 complementary to the left side of the restriction site R x , thermostable ligase and moderate temperature. Primer assembly occurs internally within several overlapping PENTAmer molecules except PENTAmer X where priming occurs at the "restriction" end of the DNA fragment in the region immediately adjacent to the adaptor sequence A (FIG. 14B).
  • Step 2 Polymerase extension reaction at an elevated temperature.
  • Step 3 Ligation of the tagged oligonucleotide PA to the 5' end of the extension product.
  • Oligonucleotide P A is complementary to the adaptor A and it is ligated only to the terminally extended molecule on the targeted PENTAmer X (FIG. 14D).
  • Step 4 Degradation of the template PENTAmer DNA library by incubation with dU-glycosylase followed by heating.
  • FIGS. 15A through 15E show a third approach. It involves four major steps.
  • Step 1 Ligation-assembly reaction using short phosphorylated oligonucleotides Pi, P 2 , P 3 complementary to the left side of the restriction site R x and the tagged oligonucleotide PA complementary to the adaptor A DNA sequence, thermostable ligase and moderate temperature. Assembly of larger oligomers from oligos Pi, P 2 , P 3 occurs internally within several overlapping PENTAmer molecules but incorporation of the tailed oligo P A occurs only at the end of the PENTAmer X (FIG. 15B)
  • Step 2 Polymerase extension reaction at elevated temperature. Priming occurs internally within several overlapping PENTAmer molecules but only extension reaction with PENTAmer X as a template results in a full size product with PA tail (sequence C) at the 5' end (FIG. 15C).
  • Step 3 Degradation of the template PENTAmer DNA library by incubation with dU-glycosylase followed by heating (FIG. 15D).
  • Step 4 PCR amplification using primers B and C (5' portion of the tagged oligo P A ) (FIG. 15E).
  • PENTAmer molecules have a single stranded form; b) the strand complementary to the primary PENTAmer is used for the selection, namely, the strand 5 'B - 3 'A (the primary PENTAmer has an opposite orientation 5 'A -> 3'B) (FIGS. 5 A and 5B); c) molecules are immobilized through a 5 '-biotin group (primer B) on the solid support (magnetic beads); and d) a fraction of dT nucleotides is replaced with dU nucleotides during preparation of the PENTAmer library
  • Conditions a) and b) are important prerequisites of protocols ##1, 2 and 3 for targeted PENTAmer amplification.
  • Factor d) allows elimination of original templates and reduces amplification of the non-specific products.
  • the first method utilizes a standard about 20-30 base long oligo-primer for the extension reaction.
  • the primer is assembled by ligation of short (i.e. octamers) phosphorylated target-specific oligonucleotides P n from a pre-synthesized oligo-library.
  • FIGS. 14 and 15 show the assembly of only three sequence-specific oligonucleotides Pi, P 2 , P , but their number can be substantially higher.
  • the third method combines into one step a ligation of the target-specific oligonucleotides P n and the adaptor- specific oligo PA- [0210]
  • the second and third selection protocols are preferable to the first protocol presented in FIGS. 13A-13E.
  • they allow an increase in the stringency of the primer-extension step.
  • polymerases are more sensitive to the mismatches within the 3 ' region of the primer and can easily tolerate mis-pairing in the central and 5 '-portion.
  • Thermostable ligases are also better at discriminating mismatches located at the 3 ' end of the oligonucleotides during their ligation.
  • primer assembly by ligation of short DNA molecules allows increase in the specificity and the selection power of the targeted amplification method due to the higher mismatch discrimination of multiple internal base positions within the priming site.
  • the fourth protocol is different in that it uses a non-immobilized DNA library and adds an additional selection step at the level of affinity capture of the ligation- selected primer-extended PENTAmer molecules (FIGS. 16A through 16E). Otherwise, it is similar to the Method 1.
  • FIGS. 16A through 16E show the fourth targeted amplification method involving five major steps. '
  • Step 1 Polymerase extension reaction with phosphorylated primer- selector P complementary to the left side of the restriction site R and Bst (heat sensitive) DNA polymerase (FIGS. 16A and 16B).
  • Priming occurs internally within several overlapping PENTAmer molecules except PENTAmer X where priming occurs at the "restriction" end of the DNA fragment in the region immediately adjacent to the adaptor sequence A.
  • Step 2 Heat inactivation of Bst DNA polymerase (FIG. 16C).
  • Step 3 Ligation of the tagged oligonucleotide PA to the 5' end of the extension product.
  • Oligonucleotide P A is complementary to the adaptor A and it is ligated only to the terminally extended molecule on the targeted PENTAmer X (FIG. 16D).
  • Step 4 Magnetic bead capture of the targeted PENTAmer X (FIG. 16E).
  • Step 5 PCR amplification using primers B and C (5' portion of the tagged oligo P A ) or B and A (FIG. 16 F).
  • e Removal of dU-containing DNA molecules
  • a skilled artisan recognizes that it would be useful to separate a desired molecule, or more than one, from an undesired molecule, or more than one.
  • a skilled artisan is aware of a variety of means to achieve this, but in the present invention it is preferred to polymerize nick translate molecules in the presence of dU nucleotides, but alternatively polymerize a desired primer extension molecule having no incorporation of dU.
  • this occurs in the absence of dU nucleotides in a reaction mixture.
  • the dU-containing molecules are then subjected to a dU glycosylase, such as AmpErase Uracil N-glycosylase (UNG) (Applied Biosystems, Foster City, CA).
  • UNG AmpErase Uracil N-glycosylase
  • UNG acts on single- or double-stranded dU-containing DNA by hydrolysis of uracil-glycosidic bonds (base excision) at dU-containing DNA sites, releasing uracil and creating an alkali-sensitive apyrimidinic site in the DNA.
  • uracil N-glycosylase can be used to cleave DNA at any position where a deoxyuridine triphosphate has been incorporated.
  • Example 6 and FIG. 5 shows 55 different loci in the bacterial genome amplified using the PENTAmer library prepared by a partial digestion of the E. coli genomic DNA with the Sau3A I restriction enzyme (Example 5), universal primer B (Table VII) and 40 E. co/z ' -specific primers (Table VII).
  • the electrophoretic profiles show a complex multi-band pattern with a maximum size of 1 kb (the PENTAmer size).
  • the PCR products have been subjected to the cycle sequencing protocol using fluorescent dye-terminators and the same primers as used for PCR and then analyzed using the MEGABASE capillary DNA sequencer.
  • the sequencing data have been analyzed by the Megabase capillary sequencing machine (Amersham; Piscataway, NJ).
  • the contribution of the adaptor DNA is very small because of two reasons: small size of the B region and the diffuse position of the "fuzzy" end with respect to the DNA priming site. If one assumes the same width of size distribution for both "fragments,” it means there are the same number of molecules within a specific size sub- interval. For example, for the interval shown on FIG. 17 by two dashed vertical lines, the total number of molecules with a correct DNA sequence is equal to 13 "molecules" originated from the "fragment” L plus 5 molecules originated from the "fragment” S, with total number 18. The number of short “fragments” within the same interval is equal to 3 giving the ratio of 0.17 for the contribution of the "bad" sequence B into the "good” signal.
  • the diffuse size distribution of the PENTAmer molecules is inherent to the nick-translation process, and it is useful. It is sufficiently narrow to allow one to control the average size of PENTAmers, and it is broad enough to minimize the effect of the B adaptor on the quality of DNA sequencing. It is clear that contribution of the B sequence can be further minimized by shortening of its size or even complete physical elimination of the terminal B sequence from the ends of amplified DNA templates.
  • the latter can be achieved by a) by a limited trimming of DNA samples after PCR with 5 ' exonuclease ( ⁇ exonuclease, or T7 gene 6 exonuclease); and/or b) by incorporation of the dU nucleotide or a ribonucleotide into the 3' portion of the B primer sequence and degradation of the B sequence using dU-glycosylase and/or alkaline hydrolysis, respectively.
  • FIG. 18 illustrates the sequencing of gaps in a genome, such as a bacterial genome, using primary PENTAmer libraries. 2. 1-2 time redundancy genomic sequencing
  • the PENTAmer walking technology can be used to sequence bacterial genomes with a minimal redundancy. For example, in a first phase the genome can be sequenced randomly with 1 time redundancy and then finished using PENTAmer library. Because the library preparation is cheap, the cost would mostly be determined by the cost of one sequence-specific oligonucleotide, which is about $ 2-3 for a 24-mer. That means that at about 600 bases obtained at each step, the oligo cost per base is going to be 0.5 cent plus additional 0.5-1 cent per base for routine sequencing operation.
  • the bacterial PENTAmer library can be diluted up to 1000 times, amplified and used for recovery DNA sequence information suggests that it is suitable for making libraries from a small amount of starting material, for example, unculturable bacteria or when there are other factors limiting the amount of DNA.
  • the PENTAmer libraries can be prepared from a complex mixture of different microorganisms.
  • the walking process will allow (with some limitations) sequence of individual genomes within a mix with other DNA.
  • FIG. 19 the fundamental nature of the present invention is illustrated in FIG. 19, wherein positional genome walking occurs by targeted PENTAmer amplification.
  • Genes are sequences of DNA in an organism's genome encoding information that is converted into various products making up a whole cell. They are expressed by the process of transcription, which involves copying the sequence of DNA into RNA. Most genes encode information to make proteins, but some encode RNAs involved in other processes. If a gene encodes a protein, its transcription product is called mRNA ("messenger" RNA). After transcription in the nucleus (where DNA is located), the mRNA must be transported into the cytoplasm for the process of translation, which converts the code of the mRNA into a sequence of amino acids to form protein.
  • mRNA messenger
  • the 3' ends of mRNA molecules are post-transcriptionally modified by addition of several adenylate residues to form the "polyA" tail.
  • This characteristic modification distinguishes gene expression products destined to make protein from other molecules in the cell, and thereby provides one means for detecting and monitoring the gene expression activities of a cell.
  • nucleic acid will generally refer to at least one molecule or strand of DNA, RNA or a derivative or mimic thereof, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g. adenine "A,” guanine “G,” thymine “T” and cytosine “C”) or RNA (e.g. A, G, uracil “U” and C).
  • nucleic acid encompass the terms “oligonucleotide” and “polynucleotide.”
  • oligonucleotide refers to at least one molecule of between about 3 and about 100 nucleobases in length.
  • polynucleotide refers to at least one molecule of greater than about 100 nucleobases in length. These definitions generally refer to at least one single- stranded molecule, but in specific embodiments will also encompass at least one additional strand that is partially, substantially or fully complementary to the at least one single-stranded molecule. Thus, a nucleic acid may encompass at least one double-stranded molecule or at least one triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a ⁇ strand of the molecule.
  • a single stranded nucleic acid may be denoted by the prefix "ss”, a double stranded nucleic acid by the prefix "ds”, and a triple stranded nucleic acid by the prefix "ts.”
  • Nucleic acid(s) that are “complementary” or “complement(s)” are those that are capable of base-pairing according to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules.
  • the term “complementary” or “complement(s)” also refers to nucleic acid(s) that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above.
  • substantially complementary refers to a nucleic acid comprising at least one sequence of consecutive nucleobases, or semiconsecutive nucleobases if one or more nucleobase moieties are not present in the molecule, are capable of hybridizing to at least one nucleic acid strand or duplex even if less than all nucleobases do not base pair with a counterpart nucleobase.
  • a "substantially complementary" nucleic acid contains at least one sequence in which about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about 80%, about 81%), about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, to about 100%, and any range therein, of the nucleobase sequence is capable of base-pairing with at least one single or double stranded nucleic acid molecule during hybridization.
  • the term “substantially complementary” refers to at least one nucleic acid that may hybridize to at least one nucleic acid strand or duplex in stringent conditions.
  • a “partly complementary” nucleic acid comprises at least one sequence that may hybridize in low stringency conditions to at least one single or double stranded nucleic acid, or contains at least one sequence in which less than about 70% of the nucleobase sequence is capable of base-pairing with at least one single or double stranded nucleic acid molecule during hybridization.
  • hybridization As used herein, “hybridization”, “hybridizes” or “capable of hybridizing” is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature.
  • the term “hybridization”, “hybridize(s)” or “capable of hybridizing” encompasses the terms “stringent condition(s)” or “high stringency” and the terms “low stringency” or “low stringency condition(s).”
  • stringent condition(s) or “high stringency” are those that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but precludes hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating at least one nucleic acid, such as a gene or nucleic acid segment thereof, or detecting at least one specific mRNA transcript or nucleic acid segment thereof, and the like.
  • Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence of formamide, tetramethylammonium chloride or other solvent(s) in the hybridization mixture. It is generally appreciated that conditions may be rendered more stringent, such as, for example, the addition of increasing amounts of formamide.
  • low stringency or “low stringency conditions”
  • non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20°C to about 50°C.
  • hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20°C to about 50°C.
  • nucleobase refers to a naturally occurring heterocyclic base, such as A, T, G, C or U ("naturally occurring nucleobase(s)"), found in at least one naturally occurring nucleic acid (i.e. DNA and RNA), and their naturally or non-naturally occurring derivatives and mimics.
  • nucleobases include purities and pyrimidines, as well as derivatives and mimics thereof, which generally can form one or more hydrogen bonds (“anneal” or “hybridize”) with at least one naturally occurring nucleobase in manner that may substitute for naturally occurring nucleobase pairing (e.g. the hydrogen bonding between A and T, G and C, and A and U).
  • nucleotide refers to a nucleoside further comprising a "backbone moiety” generally used for the covalent attachment of one or more nucleotides to another molecule or to each other to form one or more nucleic acids.
  • the "backbone moiety” in naturally occurring nucleotides typically comprises a phosphorus moiety, which is covalently attached to a 5-carbon sugar. The attachment of the backbone moiety typically occurs at either the 3'- or 5'-position of the 5-carbon sugar.
  • Restriction-enzymes recognize specific short DNA sequences four to eight nucleotides long (see Table I), and cleave the DNA at a site within this sequence.
  • restriction enzymes are used to cleave DNA molecules at sites co ⁇ esponding to various restriction-enzyme recognition sites.
  • the site may be specifically modified to allow for the initiation of the PENT reaction.
  • primers can be designed comprising nucleotides corresponding to the recognition sequences. These primers, further comprising PENT initiation sites may be ligated to the digested DNA.
  • Restriction-enzymes recognize specific short DNA sequences four to eight nucleotides long (see Table I), and cleave the DNA at a site within this sequence.
  • restriction enzymes are used to cleave cDNA molecules at sites corresponding to various restriction-enzyme recognition sites. Frequently cutting enzymes, such as the four-base cutter enzymes, are prefe ⁇ ed as this yields DNA fragments that are in the right size range for subsequent amplification reactions.
  • Some of the prefe ⁇ ed four-base cutters are Nlalll, DpnII, Sau3Al, Hsp92II, Mbol, Ndell, Bspl431, Tsp509 I, Hhal, HinPlI, Hpall, Mspl, Taq alphal, Maell or K2091.
  • primers can be designed comprising nucleotides co ⁇ esponding to the recognition sequences. If the primer sets have in addition to the restriction recognition sequence, degenerate sequences co ⁇ esponding to different combinations of nucleotide sequences, one can use the primer set to amplify DNA fragments that have been cleaved by the particular restriction enzyme.
  • the list below exemplifies the currently known restriction enzymes that may be used in the invention.
  • nucleic acid modifying enzymes listed in the following tables.
  • DNA Polymerase I Klenow Fragment, Exonuclease Minus
  • the DNA polymerase will retain 5'-3' exonuclease activity. Nevertheless, it is envisioned that the methods of the invention could be' carried out with one or more enzymes where multiple enzymes combine to carry out the function of a single DNA polymerase molecule retaining 5'-3' exonuclease activity.
  • Effective polymerases which retain 5'-3' exonuclease activity include, for example, E. coli DNA polymerase I, Taq DNA polymerase, S. pneumoniae DNA polymerase I, Tfl DNA polymerase, D. radiodurans DNA polymerase I, Tth DNA polymerase, Tth XL DNA polymerase, M.
  • tuberculosis DNA polymerase I M. thermoautotrophicum DNA polymerase I, Herpes simplex- 1 DNA polymerase, E. coli DNA polymerase I Klenow fragment, Vent DNA polymerase, thermosequenase and wild-type or modified T7 DNA polymerases.
  • the effective polymerase is E. coli DNA polymerase I, M. tuberculosis DNA polymerase I or Taq DNA polymerase.
  • the break in the substantially double stranded nucleic acid template is a gap of at least a base or nucleotide in length that comprises, or is reacted to comprise, a 3' hydroxyl group
  • the range of effective polymerases that may be used is even broader.
  • the effective polymerase may be, for example, E. coli DNA polymerase I, Taq DNA polymerase, S. pneumoniae DNA polymerase I, Tfl DNA polymerase, D. radiodurans DNA polymerase I, Tth DNA polymerase, Tth XL DNA polymerase, M. tubercidosis DNA polymerase I, M.
  • thermoautotrophicum DNA polymerase I Herpes simplex- 1 DNA polymerase, E. coli DNA polymerase I Klenow fragment, T4 DNA polymerase, vent DNA polymerase, the ⁇ nosequenase or a wild-type or modified T7 DNA polymerase.
  • the effective polymerase is E. coli DNA polymerase I, M. tuberculosis DNA polymerase I, Taq DNA polymerase or T4 DNA polymerase.
  • PENTAmer synthesis requires the use of primers which hybridize to specific sequences. Further, PENT reaction products may be useful as probes in hybridization analysis.
  • nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired.
  • Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
  • relatively high stringency conditions For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids.
  • relatively low salt and/or high temperature conditions such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C.
  • Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
  • Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature.
  • a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C.
  • Hybridization conditions can be readily manipulated depending on the desired results.
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 3 mM MgCl 2 , 1.0 mM dithiotlireitol, at temperatures between approximately 20°C to about 37°C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl 2 , at temperatures ranging from approximately 40°C to about 72°C.
  • Nucleic acids useful as templates for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et al, 1989). In certain embodiments, analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid.
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA.
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is prefe ⁇ ed.
  • Pairs of primers designed to selectively hybridize to nucleic acids are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids contain one or more mismatches with the primer sequences.
  • the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also refe ⁇ ed to as "cycles," are conducted until a sufficient amount of amplification product is produced.
  • the amplification product may be detected or quantified.
  • the detection may be perfo ⁇ ned by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals (Affymax technology).
  • PCRTM polymerase chain reaction
  • two synthetic oligonucleotide primers which are complementary to two regions of the template DNA (one for each strand) to be amplified, are added to the template DNA (that need not be pure), in the presence of excess deoxynucleotides (dNTPs) and a thermostable polymerase, such as, for example, Taq (Thermus aquaticus) DNA polymerase.
  • dNTPs deoxynucleotides
  • a thermostable polymerase such as, for example, Taq (Thermus aquaticus) DNA polymerase.
  • the target DNA is repeatedly denatured (around 90°C), annealed to the primers (typically at 50-60°C) and a daughter strand extended from the primers (72°C).
  • the daughter strands act as templates in subsequent cycles.
  • the template region between the two primers is amplified exponentially, rather than linearly.
  • a reverse transcriptase PCRTM amplification procedure may be performed to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989.
  • Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641.
  • Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Patent No. 5,882,864.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Patent Application No. PCT US87/00880, also may be used as still another amplification method in the present invention, h this method, a replicative sequence of RNA which has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which can then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[ ⁇ -thio]-triphosphates in one strand of a restriction site also may be useful in the amplification of nucleic acids in the present invention.
  • Such an amplification method is described by Walker et al. 1992, incorporated herein by reference.
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • Target specific sequences can also be detected using a cyclic probe reaction (CPR).
  • CPR cyclic probe reaction
  • a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA which is present in a sample.
  • the reaction is treated with RNase H, and the products of the probe identified as distinctive products which are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR, Kwoh et al, 1989; PCT Patent Application WO 88/10315 et al, 1989, each incorporated herein by reference).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR 3SR
  • PCT Patent Application WO 88/10315 et al, 1989 each incorporated herein by reference.
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer which has target specific sequences.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6.
  • a polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • Suitable amplification methods include “race” and “one-sided PCRTM” (Frohman, 1990; Ohara et al, 1989, each herein incorporated by reference). Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, also may be used in the amplification step of the present invention, Wu et al, 1989, incorporated herein by reference). IX. DETECTION OF NUCLEIC ACIDS
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, 1989). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid.
  • Separation of nucleic acids may also be effected by chromatographic techniques known in art. There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.
  • the amplification products are visualized.
  • a typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light.
  • the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, or another binding partner carrying a detectable moiety.
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art. See Sambrook et al, 1989.
  • U.S. Patent No. 5,279,721, incorporated by reference herein discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
  • Other methods of nucleic acid detection that may be used in the practice of the instant invention are disclosed in U.S. Patent Nos.
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al , 1989).
  • Separation by electrophoresis is based upon the differential migration through a gel according to the size and ionic charge of the molecules in an electrical field.
  • High resolution techniques normally use a gel support for the fluid phase. Examples of gels used are starch, acrylamide, agarose or mixtures of acrylamide and agarose. Frictional resistance produced by the support causes size, rather than charge alone, to become the major determinant of separation. Smaller molecules with a more negative charge will travel faster and further through the gel toward the anode of an electrophoretic cell when high voltage is applied. Similar molecules will group on the gel. They may be visualized by staining and quantitated, in relative terms, using densitometers which continuously monitor the photometric density of the resulting stain.
  • the electrolyte may be continuous (a single buffer) or discontinuous, where a sample is stacked by means of a buffer discontinuity, before it enters the running gel/ running buffer.
  • the gel may be a single concentration or gradient in which pore size decreases with migration distance.
  • SDS gel electrophoresis of proteins or electrophoresis of polynucleotides mobility depends primarily on size and is used to determined molecular weight.
  • pulse field electrophoresis two fields are applied alternately at right angles to each other to minimize diffusion mediated spread of large linear polymers.
  • Agarose gel electrophoresis facilitates the separation of DNA or RNA based upon size in a matrix composed of a highly purified form of agar. Nucleic acids tend to become oriented in an end on position in the presence of an electric field. Migration through the gel matrices occurs at a rate inversely proportional to the logic of the number of base pairs (Sambrook et al. , 1989).
  • Polyacrylamide gel electrophoresis is an analytical and separative technique in which molecules, particularly proteins, are separated by their different electrophoretic mobilities in a hydrated gel.
  • the gel suppresses convective mixing of the fluid phase through which the electrophoresis takes place and contributes molecular sieving.
  • SDS anionic detergent sodium dodecylsulphate
  • chromatographic techniques may be employed to effect separation.
  • chromatography There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography (Freifelder, 1982).
  • labeled cDNA products such as biotin or antigen can be captured with beads bearing avidin or antibody, respectively.
  • Microfluidic techniques include separation on a platform such as microcapillaries, designed by ACLARA BioSciences Inc., or the LabChipTM "liquid integrated circuits" made by Caliper Technologies Inc. These microfluidic platforms require only nanoliter volumes of sample, in contrast to the microliter volumes required by other separation technologies. Miniaturizing some of the processes involved in genetic analysis has been achieved using microfluidic devices. For example, published PCT Application No. WO 94/05414, to Northrup and White, incorporated herein by reference, reports an integrated micro-PCRTM apparatus for collection and amplification of nucleic acids from a specimen. U.S. Patent Nos.
  • micro capillary a ⁇ ays are contemplated to be used for the analysis.
  • Microcapillary a ⁇ ay electrophoresis generally involves the use of a thin capillary or channel that may or may not be filled with a particular separation medium. Electrophoresis of a sample through the capillary provides a size based separation profile for the sample. The use of microcapillary electrophoresis in size separation of nucleic acids has been reported in, for example, WooUey and Mathies, 1994. Microcapillary a ⁇ ay electrophoresis generally provides a rapid method for size-based sequencing, PCRTM product analysis and restriction fragment sizing. The high surface to volume ratio of these capillaries allows for the application of higher electric fields across the capillary without substantial thermal variation across the capillary, consequently allowing for more rapid separations.
  • Furthennore when combined with confocal imaging methods, these methods provide sensitivity in the range of attomoles, which is comparable to the sensitivity of radioactive sequencing methods.
  • Microfabrication of microfluidic devices including microcapillary electrophoretic devices has been discussed in detail in, for example, Jacobsen et al, 1994; Effenhauser et al, 1994; Harrison et al, 1993; Effenhauser et al, 1993; Manz et al, 1992; and U.S. Patent No. 5,904,824, here incorporated by reference.
  • these methods comprise photolithographic etching of micron scale channels on a silica, silicon or other crystalline substrate or chip, and can be readily adapted for use in the present invention.
  • the capillary a ⁇ ays may be fabricated from the same polymeric materials described for the fabrication of the body of the device, using the injection molding techniques described herein.
  • Tsuda et al, 1990 describes rectangular capillaries, an alternative to the cylindrical capillary glass tubes.
  • Some advantages of these systems are their efficient heat dissipation due to the large height-to-width ratio and, hence, their high surface-to-volume ratio and their high detection sensitivity for optical on-column detection modes.
  • These flat separation channels have the ability to perform two-dimensional separations, with one force being applied across the separation channel, and with the sample zones detected by the use of a multi-channel array detector.
  • the capillaries e.g., fused silica capillaries or channels etched, machined or molded into planar substrates, are filled with an appropriate separation/sieving matrix.
  • sieving matrices include, e.g., hydroxyethyl cellulose, polyacrylamide, agarose and the like.
  • the specific gel matrix, running buffers and running conditions are selected to maximize the separation characteristics of the particular application, e.g., the size of the nucleic acid fragments, the required resolution, and the presence of native or undenatured nucleic acid molecules.
  • running buffers may include denaturants, chaotropic agents such as urea or the like, to denature nucleic acids in the sample.
  • Mass spectrometry provides a means of "weighing" individual molecules by ionizing the molecules in vacuo and making them “fly” by volatilization. Under the influence of combinations of electric and magnetic fields, the ions follow trajectories depending on their individual mass (m) and charge (z). For low molecular weight molecules, mass spectrometry has been part of the routine physical-organic repertoire for analysis and characterization of organic molecules by the determination of the mass of the parent molecular ion. In addition, by a ⁇ anging collisions of this parent molecular ion with other particles (e.g., argon atoms), the molecular ion is fragmented forming secondary ions by the so-called collision induced dissociation (CID).
  • CID collision induced dissociation
  • ES mass spectrometry was introduced by Fenn, 1984; PCT Application No. WO 90/14148 and its applications are summarized in review articles, for example, Smith 1990 and Ardrey, 1992.
  • a mass analyzer a quadrupole is most frequently used. The determination of molecular weights in femtomole amounts of sample is very accurate due to the presence of multiple ion peaks which all could be used for the mass calculation.
  • MALDI mass spectrometry in contrast, can be particularly attractive when a time-of-flight (TOF) configuration is used as a mass analyzer.
  • TOF time-of-flight
  • the MALDI-TOF mass spectrometry has been introduced by Hillenkamp 1990. Since, in most cases, no multiple molecular ion peaks are produced with this technique, the mass spectra, in principle, look simpler compared to ES mass spectrometry. DNA molecules up to a molecular weight of 410,000 daltons could be desorbed and volatilized (Williams, 1989).
  • FET fluorescence energy transfer
  • the excited-state energy of the donor fluorophore is transferred by a resonance dipole-induced dipole interaction to the neighboring acceptor. This results in quenching of donor fluorescence.
  • the acceptor is also a fluorophore, the intensity of its fluorescence may be enhanced.
  • the efficiency of energy transfer is highly dependent on the distance between the donor and acceptor, and equations predicting these relationships have been developed by Forster, 1948.
  • the distance between donor and acceptor dyes at which energy transfer efficiency is 50% is refe ⁇ ed to as the Forster distance (Ro).
  • Other mechanisms of fluorescence quenching are also known including, for example, charge transfer and collisional quenching.
  • Higuchi (1992) discloses methods for detecting DNA amplification in real-time by monitoring increased fluorescence of ethidium bromide as it binds to double-stranded DNA. The sensitivity of this method is limited because binding of the ethidium bromide is not target specific and background amplification products are also detected.
  • Lee, 1993 discloses a realtime detection method in which a doubly-labeled detector probe is cleaved in a target amplification-specific mamier during PCRTM.
  • the detector probe is hybridized downstream of the amplification primer so that the 5'-3' exonuclease activity of Taq polymerase digests the detector probe, separating two fluorescent dyes which form an energy transfer pair. Fluorescence intensity increases as the probe is cleaved.
  • Published PCT application WO 96/21144 discloses continuous fluorometric assays in which enzyme-mediated cleavage of nucleic acids results in increased fluorescence. Fluorescence energy transfer is suggested for use in the methods, but only in the context of a method employing a single fluorescent label which is quenched by hybridization to the target.
  • Signal primers or detector probes which hybridize to the target sequence downstream of the hybridization site of the amplification primers have been described for use in detection of nucleic acid amplification (U.S. Pat. No. 5,547,861).
  • the signal primer is extended by the polymerase in a manner similar to extension of the amplification primers. Extension of the amplification primer displaces the extension product of the signal primer in a target amplification-dependent manner, producing a double-stranded secondary amplification product which may be detected as an indication of target amplification.
  • the secondary amplification products generated from signal primers may be detected by means of a variety of labels and reporter groups, restriction sites in the signal primer which are cleaved to produce fragments of a characteristic size, capture groups, and structural features such as triple helices and recognition sites for double-stranded DNA binding proteins.
  • FITCytetramethylrhodamine isothiocyanate FITC/Texas RedTM.
  • Molecular Probes FITC/N-hydroxysuccinimidyl 1-pyrenebutyrate (PYB), FITC/eosin isothiocyanate (EITC), N-hydroxysuccinimidyl 1-pyrenesulfonate (PYS)/FITC, FITC/Rhodamine X, FITC/tetramethylrhodamine (TAMRA), and others.
  • the selection of a particular donor/acceptor fluorophore pair is not critical.
  • DABYL dimethyl aminophenylazo benzoic acid
  • EDANS 5-(2'- aminoethyl) aminonaphthalene
  • Any dye pair which produces fluorescence quenching in the detector nucleic acids of the invention are suitable for use in the methods of the invention, regardless of the mechanism by which quenching occurs.
  • Terminal and internal labeling methods are both known in the art and maybe routinely used to link the donor and acceptor dyes at their respective sites in the detector nucleic acid.
  • DNA a ⁇ ays and gene chip technology provides a means of rapidly screening a large number of DNA samples for their ability to hybridize to a variety of single stranded DNA probes immobilized on a solid substrate.
  • chip-based DNA technologies such as those described by Hacia et al, (1996) and Shoemaker et al. (1996). These teclmiques involve quantitative methods for analyzing large numbers of genes rapidly and accurately The technology capitalizes on the complementary binding properties of single stranded DNA to screen DNA samples by hybridization. Pease et al, 1994; Fodor et al, 1991.
  • a DNA a ⁇ ay or gene chip consists of a solid substrate upon which an array of single stranded DNA molecules have been attached.
  • the chip or array is contacted with a single stranded DNA sample which is allowed to hybridize under stringent conditions.
  • the chip or a ⁇ ay is then scanned to determine which probes have hybridized.
  • probes could include synthesized oligonucleotides, cDNA, genomic DNA, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), chromosomal markers or other constructs a person of ordinary skill would recognize as adequate to demonstrate a genetic change.
  • a variety of gene chip or DNA array formats are described in the art, for example US Patent Nos. 5,861,242 and 5,578,832 which are expressly incorporated herein by reference.
  • a means for applying the disclosed methods to the construction of such a chip or a ⁇ ay would be clear to one of ordinary skill in the art.
  • the basic structure of a gene chip or array comprises: (1) an excitation source; (2) an a ⁇ ay of probes; (3) a sampling element; (4) a detector; and (5) a signal amplification treatment system.
  • a chip may also include a support for immobilizing the probe.
  • a target nucleic acid may be tagged or labeled with a substance that emits a detectable signal; for example, luminescence.
  • the target nucleic acid may be immobilized onto the integrated microchip that also supports a phototransducer and related detection circuitry.
  • a gene probe may be immobilized onto a membrane or filter which is then attached to the microchip or to the detector surface itself.
  • the immobilized probe may be tagged or labeled with a substance that emits a detectable or altered signal when combined with the target nucleic acid.
  • the tagged or labeled species may be fluorescent, phosphorescent, or otherwise luminescent, or it may emit Raman energy or it may absorb energy.
  • the DNA probes may be directly or indirectly immobilized onto a transducer detection surface to ensure optimal contact and maximum detection.
  • the ability to directly synthesize on or attach polynucleotide probes to solid substrates is well known in the art. See U.S. Patent Nos. 5,837,832 and 5,837,860 both of which are expressly incorporated by reference. A variety of methods have been utilized to either permanently or removably attach the probes to the substrate.
  • Exemplary methods include: the immobilization of biotinylated nucleic acid molecules to avidin streptavidin coated supports (Holmstrom, 1993), the direct covalent attachment of short, 5'-phosphorylated primers to chemically modified polystyrene plates (Rasmussen, et al, 1991), or the precoating of the polystyrene or glass solid phases with poly-L-Lys or poly L-Lys, Phe, followed by the covalent attachment of either amino- or sulfhydryl-modified oligonucleotides using bi-functional crosslinking reagents. (Running, et al, 1990); Newton, et al. (1993)).
  • the probes When immobilized onto a substrate, the probes are stabilized and therefore may be used repeatedly.
  • hybridization is performed on an immobilized nucleic acid target or a probe molecule is attached to a solid surface such as nitrocellulose, nylon membrane or glass.
  • nitrocellulose membrane reinforced nitrocellulose membrane, activated quartz, activated glass, polyvinylidene difluoride (PVDF) membrane, polystyrene substrates, polyacrylamide-based substrate, other polymers such as poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals capable of forming covalent links with target molecules.
  • PVDF polyvinylidene difluoride
  • PVDF polystyrene substrates
  • polyacrylamide-based substrate other polymers such as poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals capable of forming covalent links with target molecules.
  • Binding of the probe to a selected support may be accomplished by any of several means.
  • DNA is commonly bound to glass by first silanizing the glass surface, then activating with carbodimide or glutaraldehyde.
  • Alternative procedures may use reagents such as 3-glycidoxypropyltrimethoxysilane (GOP) or aminopropyltrimethoxysilane (APTS) with DNA linked via amino linkers incorporated either at the 3' or 5' end of the molecule during DNA synthesis.
  • GOP 3-glycidoxypropyltrimethoxysilane
  • APTS aminopropyltrimethoxysilane
  • DNA may be bound directly to membranes using ultraviolet radiation. With nitrocellous membranes, the DNA probes are spotted onto the membranes.
  • a UV light source (Stratalinker, from Stratagene, La Jolla, Ca.) is used to i ⁇ adiate DNA spots and induce cross-linking.
  • An alternative method for cross-linking involves baking the spotted membranes at 80°C for two hours in vacuum.
  • Specific DNA probes may first be immobilized onto a membrane and then attached to a membrane in contact with a transducer detection surface. This method avoids binding the probe onto the transducer and may be desirable for large-scale production.
  • Membranes particularly suitable for this application include nitrocellulose membrane (e.g., from BioRad, Hercules, CA) or polyvinylidene difluoride (PVDF) (BioRad, Hercules, CA) or nylon membrane (Zeta-Probe, BioRad) or polystyrene base substrates (DNA.BINDTM Costar,
  • Amplification products must be visualized in order to confirm amplification of the target-gene(s) sequences.
  • One typical visualization method involves staining of a gel with for example, a fluorescent dye, such as ethidium bromide or Vista Green and visualization under UV light.
  • a fluorescent dye such as ethidium bromide or Vista Green
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly, using a nucleic acid probe.
  • a labeled, nucleic acid probe is brought into contact with the amplified gene(s) sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, where the other member of the binding pair carries a detectable moiety.
  • the probe incorporates a fluorescent dye or label.
  • the probe has a mass label that can be used to detect the molecule amplified.
  • Other embodiments also contemplate the use of TaqmanTM and Molecular BeaconTM probes.
  • solid-phase capture methods combined with a standard probe may be used as well.
  • PCRTM products The type of label incorporated in PCRTM products is dictated by the method used for analysis.
  • capillary electrophoresis microfluidic electrophoresis, HPLC, or LC separations, either incorporated or intercalated fluorescent dyes are used to label and detect the PCRTM products.
  • Samples are detected dynamically, in that fluorescence is quantitated as a labeled species moves past the detector. If any electrophoretic method, HPLC, or LC is used for separation, products can be detected by absorption of UV light, a property inherent to DNA and therefore not requiring addition of a label.
  • primers for the PCRTM can be labeled with a fluorophore, a chromophore or a radioisotope, or by associated enzymatic reaction.
  • Enzymatic detection involves binding an enzyme to primer, e.g., via a bioti avidin interaction, following separation of PCRTM products on a gel, then detection by chemical reaction, such as chemiluminescence generated with luminol. A fluorescent signal can be monitored dynamically.
  • Detection with a radioisotope or enzymatic reaction requires an initial separation by gel electrophoresis, followed by transfer of DNA molecules to a solid support (blot) prior to analysis. If blots are made, they can be analyzed more than once by probing, stripping the blot, and then reprobing. If PCRTM products are separated using a mass spectrometer no label is required because nucleic acids are detected directly.
  • a number of the above separation platforms can be coupled to achieve separations based on two different properties.
  • some of the PCRTM primers can be coupled with a moiety that allows affinity capture, and some primers remain unmodified.
  • Modifications can include a sugar (for binding to a lectin column), a hydrophobic group (for binding to a reverse-phase column), biotin (for binding to a streptavidin column), or an antigen (for binding to an antibody column).
  • Samples are run through an affinity chromatography column. The flow-through fraction is collected, and the bound fraction eluted (by chemical cleavage, salt elution, etc.). Each sample is then further fractionated based on a property, such as mass, to identify individual components.
  • XII SEQUENCING
  • Sanger dideoxy-termination sequencing is the means commonly employed to determine nucleotide sequence.
  • the Sanger method employs a short oligonucleotide or primer that is annealed to a single-stranded template containing the DNA to be sequenced.
  • the primer provides a 3' hydroxyl group which allows the polymerization of a chain of DNA when a polymerase enzyme and dNTPs are provided.
  • the Sanger method is an enzymatic reaction that utilizes chain-tenninating dideoxynucleotides (ddNTPs).
  • ddNTPs are chain-terminating because they lack a 3 '-hydroxyl residue which prevents fo ⁇ nation of a phosphodiester bond with a succeeding deoxyribonucleotide (dNTP).
  • dNTP deoxyribonucleotide
  • a small amount of one ddNTP is included with the four conventional dNTPs in a polymerization reaction. Polymerization or DNA synthesis is catalyzed by a DNA polymerase. There is competition between extension of the chain by incorporation of the conventional dNTPs and te ⁇ nination of the chain by incorporation of a ddNTP.
  • SequenaseTM version 2.0 is a genetically engineered form of the T7 polymerase which completely lacks 3' to 5' exonuclease activity. SequenaseTM has a very high processivity and high rate of polymerization. It can efficiently incorporate nucleotide analogs such as dITP and 7-deaza- dGTP which are used to resolve regions of compression in sequencing gels. In regions of DNA containing a high G+C content, Hoogsteen bond formation can occur which leads to compressions in the DNA. These compressions result in aberrant migration patterns of oligonucleotide strands on sequencing gels. Because these base analogs pair weakly with conventional nucleotides, intrastrand secondary structures during electrophoresis are alleviated. In contrast, Klenow does not incorporate these analogs as efficiently.
  • Taq DNA polymerase is a thermostable enzyme which works efficiently at 70-75°C.
  • the ability to catalyze DNA synthesis at elevated temperature makes Taq polymerase useful for sequencing templates which have extensive secondary structures at 37°C (the standard temperature used for Klenow and SequenaseTM reactions).
  • Taq polymerase like SequenaseTM, has a high degree of processivity and like Sequenase 2.0, it lacks 3' to 5' nuclease activity.
  • the thermal stability of Taq and related enzymes provides an advantage over T7 polymerase (and all mutants thereof) in that these thermally stable enzymes can be used for cycle sequencing which amplifies the DNA during the sequencing reaction, thus allowing sequencing to be performed on smaller amounts of DNA.
  • Optimization of the use of Taq in the standard Sanger Method has focused on modifying Taq to eliminate the intrinsic 5'-3' exonuclease activity and to increase its ability to incorporate ddNTPs to reduce inco ⁇ ect termination due to secondary structure in the single-stranded template DNA (EP 0 655 506 Bl).
  • the introduction of fluorescently labeled nucleotides has further allowed the introduction of automated sequencing which further increases processivity.
  • Immobilization of the DNA may be achieved by a variety of methods involving either non-covalent or covalent interactions between the immobilized DNA comprising an anchorable moiety and an anchor, hi a prefe ⁇ ed embodiment of the invention, immobilization consists of the non-covalent coating of a solid phase with streptavidin or avidin and the subsequent immobilization of a biotinylated polynucleotide (Holmstrom, 1993).
  • immobilization may occur by precoating a polystyrene or glass solid phase with poly-L-Lys or poly L-Lys, Phe, followed by the covalent attachment of either amino- or sulfhydryl-modified polynucleotides using bifunctional crosslinking reagents (Running, 1990 and Newton, 1993).
  • Immobilization may also take place by the direct covalent attachment of short, 5'-phosphorylated primers to chemically modified polystyrene plates ("Covalink” plates, Nunc) Rasmussen, (1991).
  • the covalent bond between the modified oligonucleotide and the solid phase surface is introduced by condensation with a water-soluble carbodiimide. This method facilitates a predominantly 5'-attachment of the oligonucleotides via their 5'- phosphates.
  • Nikiforov et al. (U.S. Patent 5610287 incorporated herein by reference) describes a method of non-covalently immobilizing nucleic acid molecules in the presence of a salt or cationic detergent on a hydrophilic polystyrene solid support containing a hydrophilic moiety or on a glass solid support.
  • the support is contacted with a solution having a pH of about 6 to about 8 containing the synthetic nucleic acid and a cationic detergent or salt.
  • the support containing the immobilized nucleic acid may be washed with an aqueous solution containing a non-ionic detergent without removing the attached molecules.
  • Gathering data from the various analysis operations will typically be carried out using methods known in the art. For example, microcapillary arrays may be scanned using lasers to excite fluorescently labeled targets that have hybridized to regions of probe arrays, which can then be imaged using charged coupled devices ("CCDs") for a wide field scanning of the array.
  • CCDs charged coupled devices
  • another particularly useful method for gathering data from the arrays is through the use of laser confocal microscopy which combines the ease and speed of a readily automated process with high resolution detection. Scanning devices of this kind are described in U.S. Patent Nos. 5,143,854 and 5,424,186.
  • the data will typically be reported to a data analysis operation.
  • the data obtained by a reader from the device will typically be analyzed using a digital computer.
  • the computer will be appropriately programmed for receipt and storage of the data from the device, as well as for analysis and reporting of the data gathered, i.e., interpreting fluorescence data to determine the sequence of hybridizing probes, normalization of background and single base mismatch hybridizations, ordering of sequence data in SBH applications, and the like, as described in, e.g., U.S. Patent Nos. 4,683,194; 5,599,668; and 5,843,651, each of which is incorporated herein by reference.
  • plant refers to any type of plant.
  • the inventors have provided below an exemplary description of some plants that may be used with the invention. However, the list is not in any way limiting, as other types of plants will be known to those of skill in the art and could be used with the invention.
  • a common class of plants exploited in agriculture are vegetable crops, including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e.g., head, leaf, romaine), bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brussels sprouts, cabbage, cardoni, carrots, napa, cauliflower, okra, onions, celery, parsley, chick peas, parsnips, chicory, Chinese cabbage, peppers, collards, potatoes, cucumber plants (marrows, cucumbers), pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant, salsify, escarole, shallots, endive, garlic, spinach, green onions, squash, greens, beet (sugar beet and fodder beet), sweet potatoes, swiss chard, horserad
  • fruit and vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, quince almonds, chestnuts, filberts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, cranberries, currants, loganbe ⁇ ies, raspbe ⁇ ies, strawberries, blackbe ⁇ ies, grapes, avocados, bananas, kiwi, persimmons, pomegranate, pineapple, tropical fruits, pomes, melon, mango, papaya, and lychee.
  • fruit and vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, quince almonds, chestnuts, filberts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, cranberries, currants, loganbe ⁇ ies, raspbe ⁇ ies, strawberries, blackbe ⁇ ies, grapes, avocados, bananas,
  • plants include bedding plants such as flowers, cactus, succulents and ornamental plants, as well as trees such as forest (broad-leaved trees and evergreens, such as conifers), fruit, ornamental, and nut-bearing trees, as well as shrubs and other nursery stock.
  • bedding plants such as flowers, cactus, succulents and ornamental plants, as well as trees such as forest (broad-leaved trees and evergreens, such as conifers), fruit, ornamental, and nut-bearing trees, as well as shrubs and other nursery stock.
  • animal refers to any type of animal.
  • the inventors have provided below an exemplary description of some animals that may be used with the invention. However, the list is not in any way limiting, as other types of animals will be known to those of skill in the art and could be used with the invention.
  • the term animal is expressly construed to include humans.
  • Animals of commercial relevance specifically include domesticated species including companion and agricultural species.
  • Bacteria is herein defined as a unicellular prokaryote. Examples include, but are not limited to, the 83 or more distinct serotypes of pneumococci, streptococci such as S. pyogenes, S. agalactiae, S. equi, S. canis, S. bovis, S. equinus, S. anginosus, S. sanguis, S. salivarius, S. mitis, S.
  • mutans other viridans streptococci, peptostreptococci, other related species of streptococci, enterococci such as Enterococcus faecalis, Enterococcus faecium, Staphylococci, such as Staphylococcus epidermidis, Staphylococcus aureus, Hemophilus influenzae, pseudomonas species such as Pseudomonas aeruginosa, Pseudomonas pseudomallei, Pseudomonas mallei, brucellas such as Brucella melitensis, Brucella suis, Brucella abortus, Bordetella pertussis, Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis, Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium pseudotuberculosis, Cory
  • Corynebacterium urealyticum, Corynebacterium hemolyticum, Corynebacterium equi, etc. Listeria monocytogenes, Nocordia asteroides, Bacteroides species, Actinomycetes species, Treponema pallidum, Leptospirosa species and related organisms.
  • the invention may also be useful for determining genomic sequences of gram negative bacteria such as Klebsiella pneumoniae, Escherichia coli, Serratia species, Acinetobacter, Francisella tularensis, Enterobacter species, Bacteriodes and like.
  • bacteria species include Bacteroides forsythus, Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens, Actinobacillus actinomycetemcomitana, Actinornyces, A. viscosus, A. naeslundii, Bacteroides forsythus, Streptococcus intermedius, Campylobacier rectus and Campylobacter jejuni, Peptostreptococcus, Eikenella corrondens, P. anaerobius, Eubacterium, P. micros, E. alactolyticum, E. brachy, Fusobacterium, F. alocis, F.
  • nucleatum nucleatum
  • Porphyromonas gingivalis Prevotella
  • P. intermedia P. nigrescens
  • Selenomonas sproda Selenomonas sproda
  • Treponema T. denticola, and T. socranskii.
  • Campylobacter species such as Cryptosporidium, Giardia, Leptospira, Pasteurella, Proteus, Shigella, Vibrio species, such as
  • Vibrio cholerae V. alginolyticus, V. fluvialis, V. mimicus, V. parahaemolyticus, V. vulnificus and other Vibrio spp., Salmonella typhimurium, S. typhi, Proteus sp., Yersinia enterocolitica, Vibrio parahaemo-lyticus, Acinetobacter calcoaceticus, Aeromonas hydrophila, A. sobria, A. caviae, C.
  • Bacterial plant pathogens include species of Agrobacteria (e.g., Agaricus bisporus (Lange) Imbach or Agrobacterium tumefaciens), Clavibacter, Corynebacterium, Erwinia (e.g., Erwinia carotovora subsp. Carotovora), Pseudomonas (e.g., Pseudomonas tolaasii Paine, Pseudomonas solanacearum, Pseudomonas syringae pv.) and Xanthomonas (e.g., Xanthomonas campestris pv. Malvacearum).
  • Agrobacteria e.g., Agaricus bisporus (Lange) Imbach or Agrobacterium tumefaciens
  • Clavibacter e.g., Corynebacterium
  • Erwinia e.g.
  • primary genomic PENTAmer library is defined as library produced from complete or partial restriction digest after ligation of nick- translation adaptor A from which a time-controlled nick-translation is performed, followed by ligation of nick-attaching adaptor B to the 3 '-terminus of synthesized PENT product.
  • Primary genomic libraries are highly representative since no amplification bias has been imposed on them.
  • This example describes a protocol for preparation of primary PENTAmer library from E. coli genomic DNA with upstream nick-translation BamB I compatible adaptor A and downstream nick-attaching adaptor B having randomized bases at the strand used to direct ligation at the 3 ' end of nick-translated PENT molecules.
  • Genomic DNA from E. coli MG-1655 is prepared by standard procedure . Ten micrograms of DNA are digested at 37°C for 4 hours with 120 units of BamB I restriction enzyme (NEB) in total volume of 150 ⁇ l. The sample is split into two tubes, diluted twice with water, supplemented with 1 x Shrimp Alkaline Phosphatase (SAP) buffer (Roche; Nutley, NJ), and the DNA is dephosphorylated with 10 units of SAP (Roche; Nutley, NJ) for 20 min at 37°C.
  • SAP SAP
  • SAP is heat-inactivated for 15 min at 65°C and DNA is purified by extraction with equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) followed by precipitation with ethanol.
  • Digested DNA is dissolved in 50 ⁇ l of 10 mM Tris-HCl, pH 7.5.
  • the sample is mixed with 3 pmoles of pre-assembled BamB I nick- translation adaptor (adaptor A3 consisting of primers 11, 12, and 13), and ligation is carried out overnight at 16°C with 1200 units of T4 ligase (NEB) in 60 ⁇ l volume.
  • the sample is extracted with equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), supplemented with 1/4 volume of QF buffer (final concentrations of 240 mM NaCl, 3 % isopropanol, and 10 mM Tris-HCl, pH 8.5) in a volume of 400 ⁇ l and centrifuged at 200 x g to a volume of approximately 100 ⁇ l.
  • the sample is washed 3 times with 400 ml of TE-L buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5) at 200 x g and concentrated to a final volume of 80 ⁇ l.
  • the purified sample is subjected to nick-translation with 20 units of wild type Taq polymerase in lx Perkin Elmer (Norwalk, CT) PCR buffer buffer II containing 2 mM MgCl 2 and 200 mM of each dNTP for 5 min at 50°C.
  • the reaction is stopped by addition of 5 ⁇ l of 0.5 M EDTA pH 8.0, and products are analyzed on 6% TBE-urea gel (Novex; San Diego, CA) after staining with Sybr Gold.
  • an oligonucleotide complementary to the template strand spanning the entire adaptor sequence (primer 15) is added at a final concentration of 0.8 mM, and the sample is denatured by boiling at 100°C for 3 min and cooling on ice for 5 min.
  • Adaptor Bl is ligated to the single-stranded library of PENT molecules bound to magnetic beads.
  • Adaptor Bl consists of two oligonucleotides: one is 5'- phosphorylated and 3 '-blocked (primer 16); and a second is its complement, which has a 3'- extension of four random bases and is also 3 '-blocked (primer 17). The latter oligonucleotide will anneal and direct the phosphorylated adaptor strand to the free 3 '-end of single-stranded genomic PENT library molecules.
  • the library DNA from the previous step is mixed with 40 pmoles of each adaptor Bl oligonucleotide (primers 16 and 17) in lx T4 ligase buffer and 1200 units of T4 ligase (NEB) in final volume of 30 ⁇ l. Ligation is performed at room temperature for 1 hour on an end-to-end rotary shaker to keep the beads in suspension. Beads are bound to magnet, washed with 2 x 100 ⁇ l each of 1 x BW buffer and TE-L buffer and nonbiotinylated DNA molecules are removed by incubating the beads in 100 ⁇ l of 0.1 N NaOH for 5 min at room temperature.
  • Beads are neutralized by washing with 5 x 100 ⁇ l of TE-L buffer, resuspended in 100 ⁇ l of storage buffer (SB buffer, containing 0.5 M NaCl, 10 mM Tris-HCl, 10 mM EDTA, pH 7.5) and stored at 4°C.
  • SB buffer containing 0.5 M NaCl, 10 mM Tris-HCl, 10 mM EDTA, pH 7.5
  • FIG. 20 shows analysis of 5 selected random sequences in the E. coli genome adjacent to BamB I sites to assess the quality and representativity of the library.
  • One microliter of library beads diluted 10 x in water (approximately 0.1 % of the total library DNA) are used as template in PCR amplification reactions with universal adaptor B 1 primer (primer 18) and 5 specific E. coli primers adjacent to BamB I sites.
  • a negative control with adaptor Bl primer alone and a positive control with adaptor Bl and adaptor A3 primers are also included.
  • After initial denaturing at 95°C for 1 min, 30 cycles of 94°C for 10 sec and 68°C for 75 sec are ca ⁇ ied out.
  • Secondary library in the following examples is defined as a library derived from primary genomic PENTAmer library by either exponential or linear amplification, which is primarily used as template for selection by ligation and/or extension directed from adaptor A toward adaptor B and thus for the purpose of this application is the strand complementary to the PENT (nick-translation) strand of the primary library form which it is derived. Secondary libraries are potentially biased in representation of genomic sequences.
  • This example describes the preparation of secondary library derived by PCR amplification of the primary PENTAmer E. coli BamB I library described in Example 1.
  • the library is diluted and amplified by PCR in the presence of dUTP and biotinylated B 1 adaptor oligonucleotide. Biotinylated dU containing strands are captured to magnetic streptavidin beads. Finally, to prevent the free 3 'ends from self-priming during primer extension reactions, 3 '-ends are blocked by transfer of dideoxy adenosine with terminal transferase.
  • the library is used as template for selection by assembly, ligation, and extension of contigs of short oligonucleotides at specific positions or for direct primer extension of kernel sequences.
  • the sample is diluted to 300 ⁇ l with TE-L buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5), supplemented with % volume of QF buffer (final concentrations of 240 mM NaCl, 3 % isopropanol, and 10 mM Tris-HCl, pH 8.5) and centrifuged at 200 x g in Microcon YM-100 (Millipore; Bedford, MA) filter to a volume of 100 ⁇ l. The sample is then washed 2 times with 400 ⁇ l of TE-L buffer at 200 x g and concentrated to a final volume of 120 ⁇ l.
  • TE-L buffer 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5
  • QF buffer final concentrations of 240 mM NaCl, 3 % isopropanol, and 10 mM Tris-HCl, pH 8.5
  • Three hundred micrograms of streptavidin-coated Dynabeads M-280 are prewashed with TE-L buffer and resuspended in 2x BW buffer (20 mM Tris-HCl, 2 mM EDTA, 2 M NaCl, pH 7.5).
  • the DNA sample is mixed with equal volume of beads suspension in 2x BW buffer and placed on rotary shaker for 1 hr at room temperature.
  • the beads are bound to magnet and washed with 3 x 100 ⁇ l each of 1 x BW buffer and TE-L buffer.
  • Non-biotinylated DNA is removed by incubating the beads in 100 ⁇ l of 0.1 N NaOH for 5 min at room temperature. Beads are neutralized by washing with 5 x 100 ⁇ l of TE-L buffer and then resuspended in 20 ml of water.
  • the beads are supplemented with lx terminal transferase buffer (Roche; Nutley, NJ), 0.25 mM CoCl 2 , 0.1 mM ddATP, and 200 units of terminal transferase (NEB) in a final volume of 50 ⁇ l and reaction is carried out at 37°C for 30 min.
  • Beads are washed with 2 x 100 ⁇ l each of TE-L buffer and 1 x BW buffer, resuspended in 50 ⁇ l of SB buffer (0.5 M NaCl, 10 mM Tris-HCl, 10 mM EDTA, pH 7.5) and stored at 4°C.
  • This example describes the assembly of contigs of 5 or 8 nonamer oligonucleotides at specific E. coli kernel sequence adjacent to BamB I restriction site by using thermo-stable ligase and secondary E. coli genomic BamBl PENTAmer library described in Example 2 as template.
  • Oligonucleotides 1, 2, 3, 4, and 5 annealing at the selected kernel as contig are mixed at final concentration of 10 nM each, except oligonucleotide 5, at 50 nM.
  • Oligonucleotide 1 is complementary in its twelve 3 '-terminal bases to adaptor A3 sequence immediately upstream from the BamB I restriction site and has an unique 5' extension of 23 bases used as PCR priming site.
  • Oligonucleotide 5 is complementary in its nine 5 '-terminal bases to the sequence being selected and has a unique 3 '-extension of 23 bases used as second priming site for PCR. All oligonucleotides except oligonucleotide 1 are 5 '-phosphorylated.
  • Oligonucleotides 1, 2, 3, 4, 5A, 6, 7 and 8 annealing at the selected kernel as contig are mixed at final concentration of 10 nM each except oligonucleotides 5 A and 8, at 50 nM.
  • Oligonucleotide 1 is complementary in its twelve 3'- terminal bases to adaptor A3 sequence immediately upstream from the BamB I restriction site and has a unique 5 ' extension of 23 bases used as PCR priming site.
  • Oligonucleotide 8 is complementary in its nine 5 '-terminal bases to the sequence being selected and has a unique 3 '-extension (identical to the extension of oligonucleotide 5) of 23 bases used as second priming site for PCR. All oligonucleotides except oligonucleotide 1 are 5 '-phosphorylated.
  • Beads are bound to magnet and supernatants neutralized with 10 ml of 0.2 N HCl and 3 ⁇ l of 1 M Tris-HCl, pH 8.0. Samples are diluted to 100 ⁇ l with water, split in 2 aliquots of 50 ⁇ l and one aliquot is treated with 1 unit of heat-labile uracil-DNA glycosylase (UDG, Roche; Nutley, NJ) for 2 hours at 20°C.
  • UDG heat-labile uracil-DNA glycosylase
  • UDG is inactivated for 10 min at 95°C and 1 ⁇ l of 3-fold diluted aliquot of each sample is used as template for PCR with primer identical to the unique 5 ' extension of oligonucleotide 1 (primer 9) and primer complementary to the unique 3' extension of oligonucleotides 5 and 8 (primer 10).
  • FIG. 22 shows analysis of 10 ⁇ l aliquots of the PCR reactions by electrophoresis on 10% TBE acrylamide gel (Novex; San Diego, CA) after staining with Sybr Gold onBio-Rad (Hercules, CA) Fluor S Multilmager. Both 5 oligonucleotide and 8 oligonucleotide contigs were assembled as evidenced by 94 bp and 121 bp amplicons obtained by PCR respectively.
  • This example demonstrates that contigs of short oligonucleotides can be successfully assembled at specific kernel positions using secondary E. coli PENTAmer library as template. Assembled contigs are stable upon washing in low salt buffer (TE-L) and can be extended with DNA polymerase at high temperature as shown in Example 4. Selected sequences can be used for walking, sequencing, and for gap filling after destroying any residual dU-containing PENTAmer molecules with uracil DNA glycosylase.
  • TE-L low salt buffer
  • Selected sequences can be used for walking, sequencing, and for gap filling after destroying any residual dU-containing PENTAmer molecules with uracil DNA glycosylase.
  • This example describes amplification of specific E. coli PENTAmer sequence by assembly of short oligonucleotides, followed by extension and ligation of universal adaptor A oligonucleotide having unique 5 '-terminal extension used as priming site for PCR.
  • Oligonucleotides 2, 3, 4, 5 A, 6, 7 and 8 A annealing as contig at specific kernel sequence adjacent to BamB I restriction site are mixed in 1 x Tsc ligase buffer (Roche; Nutley, NJ) at final concentration of 10 nM each except oligonucleotides 5 A and 8A, at 50 nM. All oligonucleotides are 5 '-phosphorylated.
  • Four microliters of 2.5-fold diluted secondary E. coli genomic BamBl PENTAmer library beads prepared as described in Example 2 are added to the oligonucleotide mix in total volume of 100 ml. The sample is divided into 3 aliquots.
  • Tcs DNA ligase (Roche; Nutley, NJ) are added to tube #1 and tube # 2 whereas tube # 3 (control) receives 1.5 ⁇ l of 1 x Tsc ligase buffer. Incubation is carried out at 45°C for 2 hours. Beads are washed 2 times with 50 ml each of 2x BW buffer and TE-L buffer and resuspended in 5 ⁇ l of water.
  • Samples are then supplemented with 1 x ThermoPol buffer (NEB), 10 mM MgCl 2 , 5 units of Bst DNA polymerase (NEB) and 0.2 mM of each dNTP in final volume of 60 ml and extension reaction is ca ⁇ ied out at 55°C for 3 min. Reactions are stopped by addition of 1 ml of 0.5M EDTA, pH 8.0 and beads are washed with 2 x 50 ⁇ l of 2x BW buffer, 2 x 50 ⁇ l of TE-L buffer and 50 ⁇ l of water. Beads are then resuspended in 25 ⁇ l of water.
  • Samples are supplemented with 1 x Tsc ligase buffer (Roche; Nutley, NJ) and 10 nM of oligonucleotide 1 (Table VII) in final volume of 30 ⁇ l.
  • Oligonucleotide 1 is complementary in its twelve 3 '-terminal bases to adaptor A3 sequence adjacent to the assembled contig and has an unique 5 ' extension of 23 bases used later as PCR priming site.
  • Five units of Tsc DNA ligase (Roche; Nutley, NJ) are added to samples #1 and # 3 whereas sample #2 receives 1 ⁇ l of 1 x Tsc ligase buffer. Ligation is carried out at 45°C for 1 hour.
  • Beads are washed sequentially with 2 x 50 ⁇ il of 2x BW buffer, 2 x 50 ⁇ l TE-L buffer, 50 ⁇ l of water, 2x 50 ⁇ l of 2x BW buffer, and 50 ⁇ l of TE-L buffer.
  • Non-biotinylated DNA is eluted with 20 ⁇ l of 0.1 N NaOH for 3 min at 37°C. Beads are removed on magnet and supernatant is neutralized with 10 ⁇ l of 0.2 N HCl and 3 ⁇ l of 1 M Tris-HCl, pH 8.0.
  • Samples are diluted to 100 ⁇ l with water, split into two aliquots of 50 ⁇ l and one half treated with 1 unit of heat-labile uracil-DNA-glycosylase (UDG, Roche; Nutley, NJ) for 2 hours at 20°C.
  • UDG heat-labile uracil-DNA-glycosylase
  • UDG is inactivated for 10 min at 95°C and 1 ⁇ l of 3-fold diluted aliquot of each sample is used as template for PCR.
  • Amplification is performed with primer identical to the unique 5' extension of oligonucleotide 1 (primer 9) or kernel primer adjacent to the Bam B I site of the selected PENTAmer and universal adaptor Bl primer (primer 18).
  • FIG. 23 shows analysis of 12 ⁇ l aliquots of the PCR reactions by electrophoresis on 10% TBE acrylamide gel (Novex; San Diego, CA) after staining with Sybr Gold performed on Bio-Rad (Hercules, CA) Fluor S Multilmager.
  • PCR amplification with both sets of primers from samples which have the contig of 9-mer oligonucleotides ligated produced a 1 Kb amplicon co ⁇ esponding to the specific PENTAmer (lanes 1, 3, and 9).
  • the control (tube # 3) in which short oligos are present but no ligase is added does not have the amplicon, indicating that no extension from short oligos occurs in the absence of ligation (lanes 5 and 13).
  • the sample which did not have adaptor A tailed oligonucleotide ligated (tube# 2) is negative when probed by PCR with the tail primer 9 (lane 11). This validates the specificity of the second ligation step.
  • non-specific PENTAmers are amplified indicating release of some biotinylated strands by NaOH treatment (lanes 2, 4, 6, 10, 12, and 14).
  • This Example describes preparation of primary PENTAmer library from E. coli genomic DNA using partial digest with frequently cutting enzyme. As shown in the following examples, this library can be used for filling gaps and de novo sequencing of genomes having the complexity of an average bacterial genome.
  • Electrophoresis in the forward direction is performed at 6 V /cm in interrupted mode (60 sec on, 5 sec off) for 1.5 hours.
  • Section of the gel containing a lane of standards and a lane of the DNA sample is excised, stained with Sybr Gold and bands are visualized on Dark Reader Blue Light Transilluminator (Clare Chemical Research). Region of the gel containing DNA molecules smaller than 2 Kb is cut out and removed. The remaining portion of the stained slice is aligned back with the unstained gel and used as a landmark for cutting and removing of the fraction containing DNA fragments bellow 2 Kb.
  • the unstained gel is then run in reverse direction in interrupted field of 6 V/cm (60 sec on, 5 sec off) for 85% of the forward time.
  • the sample is extracted with equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), mixed with 1/4 vol of QF buffer (final concentrations of 240 mM NaCl, 3% isopropanol, and 10 mM Tris-HCl, pH 8.5) in a volume of 400 ⁇ l and centrifuged at 200 - x g to a volume of approximately 100 ⁇ l on Microcon YM-100. The sample is washed 3 times with 400 ⁇ l of TE-L buffer at 200 x g and concentrated to a final volume of 135 ⁇ l.
  • QF buffer final concentrations of 240 mM NaCl, 3% isopropanol, and 10 mM Tris-HCl, pH 8.5
  • the purified sample is subjected to nick-translation with 38 units of wild type Taq polymerase in lx Perkin Elmer (Norwalk, CT) PCR buffer buffer II containing 4 mM MgCl 2 and 200 mM of each dNTP in final volume of 240 ⁇ l for 5 min at 50°C. Reaction is stopped by addition of 6 ⁇ l of 0.5 M EDTA pH 8.0 and products are analyzed on 6% TBE- urea gel (Novex; San Diego, CA) after staining with Sybr Gold.
  • the sample is supplemented with blocking oligonucleotide complementary to the nick-translation template strand adaptor sequence (primer 15) at a final concentration of 1 mM, denatured by boiling at 100°C for 3 min, and cooled on ice for 5 min. Twelve hundred micrograms of streptavidin coated Dynabeads M-280 (Dynal) are prewashed with TE-L buffer and resuspended in 2x BW buffer (20 mM Tris-HCl, 2 mM EDTA, 2 M NaCl, pH 7.5). Denatured DNA is mixed with equal volume of beads suspension in 2x BW buffer and placed on rotary shaker for 2 hr at room temperature.
  • the beads are bound to magnet and washed with 2 x 100 ⁇ l each of 1 x BW buffer and TE-L buffer.
  • Non-biotinylated DNA is removed by incubating the beads in 100 ml of 0.1 N NaOH for 5 min at room temperature.
  • Beads are washed with 100 ⁇ l of 0.1 N NaOH, neutralized by washing with 5 x 100 ⁇ l of TE- L buffer, and resuspended in 150 ⁇ l of TE-L buffer.
  • One half of the prepared library DNA is then processed for ligation with adaptor Bl.
  • the suspension 75 ⁇ l
  • the suspension is supplemented with lx T4 ligase buffer (NEB) incubated with 50 pmoles of 3 '-blocked oligonucleotides one of which is complementary to the biotinylated adaptor A strand and has 3 '-extension of 24 bases (primer 20) to which the second oligonucleotide (primer 21) is complementary.
  • NEB lx T4 ligase buffer
  • the suspension is heated for 1 min at 60°C, cooled to room temperature and incubated for 10 min at room temperature to anneal the blocking oligonucleotides to residual free adaptor A3 molecules bound to magnetic beads. Beads are then washed with 50 ⁇ l of lx T4 ligase buffer and resuspended in 50 ⁇ l of the same buffer. Adaptor Bl is then ligated to the library DNA. The sample from the previous step is supplemented with 40 pmoles of each adaptor B oligonucleotide (primers 16 and 17) in lx T4 ligase buffer and 4000 units of T4 ligase (NEB) in final volume of 55 ⁇ l.
  • NEB T4 ligase
  • Ligation is performed at room temperature for 3 hours on end-to-end rotary shaker. Beads are bound to magnet, washed with 2 x 100 ⁇ l each of 1 x BW buffer and TE-L buffer and nonbiotinylated DNA removed by incubating the beads in 100 ⁇ l of 0.1 N NaOH for 5 min at room temperature. Beads are washed with 100 ⁇ l of 0.1 N NaOH, neutralized by washing with 5 x 100 ml of TE-L buffer, resuspended in 90 ml of SB buffer and stored at 4°C.
  • FIG. 24 shows the amplification patterns obtained with 40 representative kernel primers. The bands of different size in each lane co ⁇ espond to amplified PENTAmers having the kernel sequence at different positions relative to the nick-translation termination sites (ligated adaptor Bl).
  • PENTAmer molecules are size-fractionated and are all in the range of 1 Kb, the relative position of any kernel sequence will be shifted in individual PENT molecules originating at given Sau3A I restriction site. Thus the pattern of amplification reflects the frequency of Sau3A I sites located upstream from each kernel .
  • This example demonstrates that representative normalized primary PENTAmer library can be produced from from PENTAmer library prepared from partial Sau3A I restriction digest.
  • This example validates a direct genome walking sequencing strategy for gap filling and de novo sequencing of genomes of the complexity of E. coli from PENTAmer library prepared with frequently cutting restriction enzyme.
  • PCR amplicons are purified free of polymerase, nucleotides and primers by Qiaquick PCR purification kit (Qiagen; Valencia, CA) and are eluted in 30 ⁇ l of EB buffer (Qiagen (Valencia CA), 100 mM Tris-HCl, pH 8.5). DNA is quantitated by mixing 15 ⁇ l of serial dilutions of the purified samples with equal volume of 1 :200 diluted Pico Green reagent (Molecular Probes; Eugene, OR) in TE buffer, incubating at room temperature for 5 min and spotting 20 ⁇ l aliquots along with standard amounts of DNA (low DNA Mass Ladder, Life Technologies; Rockville, MD) on Parafilm (American National Can). DNA is quantitated on Bio-Rad (Hercules, CA) Fluor S Multilmager using the volume tool of Quantity One software (Bio Rad).
  • Cycle sequencing is performed by mixing 11 ⁇ l of DNA samples containing 55-80 ng of total DNA with 1 ⁇ l of 5 mM of each kernel primer used originally in PCR (above) and 8 ⁇ l of DYEnamic ET teminator reagent mix (Amersham Pharmacia Biotech; Piscataway, NJ) in 96 well plates in final volume of 20 ⁇ l. Amplification is performed for 30 cycles at: 94°C for 2 sec, 58°C for 15 sec, and 60°C for 75 sec. Samples are precipitated with 70 % ethanol and analyzed on MegaBACE 1000 capillary sequencing system (Amersham Pharmacia Biotech; Piscataway, NJ) under the manufacturer's protocol.
  • cycle sequencing is done using the Thermo Sequenase Cy5.5 Dye Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech; Piscataway, NJ) by mixing 24 ⁇ l of template containing 20-50 ng of DNA with 1 ⁇ l of 10 mM primer, 1 ⁇ l of each individual Cy5.5 dye-labeled ddNTP teminator, 3.5 ⁇ l of reaction buffer concentrate, and 20 units of Thermo Sequenase DNA polymerase in total volume of 31.5 ⁇ l. After initial denaturing at 94°C for 1 min, amplification is performed for 30 cycles at: 94°C for 10 sec, 58°C for 30 sec, and 72°C for 1 min. Samples are purified by DyeEx dye terminator removal kit (Qiagen; Valencia, CA) and analyzed on OpenGene sequencing system (Visible Genetics).
  • Table VIII shows a summary of the sequencing results obtained with fifty E. coli kernel primers on the MegaBACE 1000 sequence analyzer in a single run. On average read lengths of the analyzed sequences are in the order of 500 bases. A sequence is considered to be a failure if about 100 or less bases are called. At a preset threshold score of >20 using the Phred algorithm (Codon Code Corporation; Dedham, MA) which co ⁇ esponds to an e ⁇ or probability of 1%, twenty two percent of the sequences failed, whereas at a Phred value of 10 (90% accuracy), the failure rate is 20%.
  • the Phred algorithm Codon Code Corporation; Dedham, MA
  • Failure rate 22% Failure rate: 20% Failure rate: 14%
  • Average read length Average read Length Average read length 554 495 (not including 546 (not including (not including failures) failures) failures)
  • a Phred score of 20 corresponds to an e ⁇ or probability of 1%.
  • c Number of bases the Phred (Codon Code Corporation, Dedham, MA) algorithm considers above the threshold score of 10.
  • the Quality Index corresponds to the accuracy rate of the called bases.
  • a sequence is considered a failure when less than 100 bases are called.
  • This example demonstrates that an average of 88% of random genomic E. coli sequences can be amplified directly from primary PENTAmer library of partial restriction digest with frequently cutting enzyme. Read lengths are on average 250 bases for the Visible Genetics instrument and 500 for the MegaBACE instrument respectively, at accuracy level of 99%. All of the failed samples that were attempted for re-sequencing by using nested primers during cycle sequencing were successful. Due to the length variation in the termination positions of PENT products during nick-translation ("fuzzy ends"), the concentration of intervening adaptor B sequences originating from Sau 3 A sites upstream of a given kernel is apparently diluted to a point where no significant interference occurs and the read length and quality of the sequencing reactions are comparable to sequencing uniformly •sized PCR fragments.
  • the remaining portion of the stained slice is aligned back with the unstained gel and used as a landmark for cutting and removing of the fraction containing undesired small molecules.
  • the unstained gel is run in reverse direction in at 6 V/cm for 85% of the forward time.
  • the gel is stained with Sybr Gold.
  • the band PENTAmer molecules now focused in a na ⁇ ow region is excised and eluted at 5,000 x g for 15 min using Ultrafree-DA gel extraction device (Milipore).
  • Sample is diluted between 10,000 and 50,000-fold and used as template for re-amplification by PCR using individual kernel primers and universal adaptor Bl primer (primer 18).
  • FIG. 25 shows an example of two E. coli genomic sequences amplified after size fractionation. Essentially all short fragments are eliminated in the second amplifications step.
  • PCR amplified samples are purified by Qiaquick PCR purification kit (Qiagen; Valencia, CA), eluted in 30 ml of EB buffer (Qiagen; Valencia, CA) and sequenced as described in Example 6.
  • This example describes the preparation of secondary library derived from the PENTAmer E. coli BamB I library shown in Example 5.
  • the library is prepared by PCR amplification of the primary library in the presence of dUTP and biotinylated B adaptor oligonucleotide, capture of the biotinylated strand on magnetic beads and blocking of its 3 'end by transfer of dideoxy adenosine with te ⁇ ninal transferase.
  • Library DNA is further size-fractionated by RF-IDF electrophoresis.
  • Sample is loaded on preparative 0.7 % pulse-field grade agarose gel (Bio Rad) along with lKb+ ladder (Life Technologies; Rockville, MD). Electrophoresis in the forward direction is performed at 6 V /cm in interrupted mode (60 sec on, 5 sec off) for 1.4 hours.
  • a section of the gel containing a lane of standards and a lane of the DNA sample is excised, stained with Sybr Gold and bands are visualized on Dark Reader Blue Light Transilluminator (Clare Chemical Research). The DNA size region smaller than 1 Kb is cut out and removed.
  • the remaining portion of the stained slice is aligned back with the unstained gel and used as landmark for cutting and removing of the fraction containing molecules below 1 Kb in size.
  • the unstained gel is then run in reverse direction in interrupted field of 6 V/cm (60 sec on, 5 sec off) for 1.1 hour.
  • the gel is stained with Sybr Gold.
  • the bands of interest focused in sharp narrow region are cut out and recovered from the agarose using Gel Extraction kit (Qiagen; Valencia, CA) in 10 mM Tris-HCl pH 8.5.
  • library beads are supplemented with lx terminal transferase buffer (Roche; Nutley, NJ), 0.25 mM CoCl 2 , 0.1 mM ddATP, and 60 units of te ⁇ ninal transferase (NEB) in a final volume of 100 ⁇ l and reaction is carried out at 37°C for 30 min.
  • Beads are washed with 2 x 100 ⁇ l each of TE-L buffer 1 x BW buffer, resuspended in 120 ⁇ l of storage buffer (0.5 M NaCl, 10 mM Tris-HCl, 10 mM EDTA, pH 7.5) and stored at 4°C.
  • This Example describes the amplification of three E. coli sequences in multiplexed linear amplification cycling reaction from secondary dU-containing Sau3A I PENTAmer library bound to magnetic beads, prepared as described in Example 8.
  • Linear amplification is performed in the presence of 3 '-blocked oligonucleotide annealing in the region of adaptor B to prevent newly synthesized single stranded molecules from self- priming.
  • the second strand is extended by adding an excess of unblocked adaptor B primer.
  • dU- containing molecules are destroyed by treatment with uracil DNA glycosylase and the sequences enriched by multiplexed linear amplification are segregated by PCR amplification with individual kernel primers and universal adaptor Bl primer.
  • oligonucleotides specific for E. coli kernel sequences adjacent to Sau3A I restriction sites are mixed in 1 x AdvanTaq ⁇ buffer (Clontech; Palo Alto, CA) at final concentration of 40 nM each with 100 nM of 3 '-blocked oligonucleotide (primer 17), 10 mM each dNTP, 10 ml of secondary dU containing Sau3A I PENTAmer library beads (Example 8) and 1 x AdvanTaq+ hot start DNA polymerase in final volume of 60 ⁇ l. Identical control reaction is assembled which lacks DNA polymerase.
  • Adaptor Bl PCR primer (primer 18) is added at final concentration of 330 nM and two more cycles are performed at 94°C for 10 sec, and 68°C for 75 sec to fill up second strand.
  • each sample is applied as template for PCR with 200 nM of each individual kernel primer used for linear amplification and 200 nM universal adaptor Bl primer (primer 18).
  • 200 nM of each individual kernel primer used for linear amplification and 200 nM universal adaptor Bl primer (primer 18).
  • a mixture of the three primers at 80 nM each and 200 nM of universal adaptor Bl primer (primer 18) are used.
  • PCR samples are analyzed on 1% agarose gel after staining with Sybr Gold.
  • FIG. 26 shows the result of this analysis. All three sequences are amplified as full-size fragments.
  • the products of the PCR amplification are purified by Qiaquick PCR purification (Qiagen; Valencia, CA) eluted in 30 ⁇ l 10 mM Tris-HCl, pH 8.5 and aliquots containing 20-50 ng of DNA are sequenced with Thermo Sequenase Cy5.5 Dye Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech) on OpenGene sequencing system (Visible Genetics) as described in Example 6 with the same kernel primers used in linear amplification and PCR. All three sequences are confirmed.
  • Qiaquick PCR purification Qiagen; Valencia, CA
  • Thermo Sequenase Cy5.5 Dye Terminator Cycle Sequencing kit Amersham Pharmacia Biotech
  • OpenGene sequencing system Visible Genetics
  • This example describes the preparation of primary human genomic PENTAmer libraries bound to magnetic beads and their amplification with universal adaptor primers.
  • DNA fragments are size-fractionated by preparative RF-IDF in 0.75% pulse-field grade agarose (Bio-Rad; Hercules, CA) gel. Electrophoresis in forward direction is performed at 6 V /cm in interrupted mode (60 sec on, 5 sec off) for 2 hours. After cutting the section of the gel containing DNA molecules below 2 Kb, reverse field of 6 V/cm (60 sec on, 5 sec off) is applied for 1.7 hours. Bands are excised and recovered from the agarose by Gel Extraction Kit (Qiagen; Valencia, CA) in 10 mM Tris-HCl pH 8.5.
  • Samples are mixed with 1.2 pmoles (BamB I) or 6 pmoles (Sau3A I) of pre-assembled BamB I nick- translation adaptor (adaptor A3 consisting of primers 11, 12, and 13) and after heating at 65°C for 1 min ligation is ca ⁇ ied out at 20°C for 2.5 hours with 4,800 units of NEB T4 ligase (BamB I) or 11,200 units of NEB T4 ligase (Sau3A I).
  • the sample is extracted with equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), mixed with 1/4 vol of QF buffer (240 mM NaCl, 3% isopropanol, and 10 mM Tris-HCl, pH 8.5 final concentrations) in a volume of 400 ⁇ l and centrifuged at 200 x g to a volume of 100 ⁇ l in Microcon YM-100 filtration units.
  • the samples are washed 3 times with 400 ⁇ l of TE-L buffer at 200 x g and concentrated to a final volume of 65 ⁇ l (BamB I) or 120 ml (Sau3A I).
  • the purified samples are subjected to nick-translation with 19 units (BamB I) or 38 units (Sau3A I) of wild type Taq polymerase in lx Perkin Elmer (Norwalk, CT) PCR buffer buffer II containing 4 mM MgCl 2 and 200 mM of each dNTP in final volume of 120 ⁇ l (BamB I) or 240 ⁇ l (Sau3A I) for 5 min at 50°C. Reactions are stopped by addition of EDTA to a final concentration of 20 mM and products are analyzed on 6% TBE-urea gel (Novex; San Diego, CA) after staining with Sybr Gold.
  • Samples are supplemented with blocking oligonucleotide complementary to the nick-translation template strand at the region of the adaptor (primer 15) at a final concentration of 1 mM, denatured by boiling at 100°C for 3 min and cooled on ice for 5 min.
  • Eighteen hundred micrograms of streptavidin coated Dynabeads M-280 (Dynal) are prewashed with TE-L buffer and resuspended in 2x BW buffer (20 mM Tris-HCl, 2 mM EDTA, 2 M NaCl, pH 7.5).
  • Denatured DNA samples are mixed with equal volume of beads (1/3 of the total beads with BamB I and 2/3 with Sau3A I sample) in 2x BW buffer and placed on rotary shaker for 1.5 hr at room temperature.
  • the beads are bound to magnet and washed 2 x with 100 ⁇ l each of 1 x BW buffer and TE-L buffer.
  • Non-biotinylated DNA is removed by incubating the beads in 100 ml of 0.1 N NaOH for 5 min at room temperature. Beads are washed with 100 ⁇ l of 0.1 N NaOH, neutralized by washing with 5 x 100 ⁇ l of TE- L buffer, and resuspended in TE-L buffer.
  • the samples are supplemented with 40 pmoles (BamB I) or 80 pmoles (Sau3A I) of each adaptor B 1 oligonucleotide (primers 16 and 17) in lx T4 ligase buffer and 4000 units (BamB I) or 8000 units (Sau3A I) of T4 ligase (NEB) in final volume of 100 ⁇ l (BamB I) or 200 ⁇ l (Sau3A I). Ligation is performed at room temperature for 3.5 hours on end-to-end rotary shaker to keep the beads in suspension.
  • Beads are bound to magnet, washed with 2 x 100 ⁇ l each of 1 x BW buffer and TE-L buffer and nonbiotinylated DNA is removed by incubating the beads in 100 ⁇ l of 0.1 N NaOH for 5 min at room temperature. Beads are washed with 100 ⁇ l of 0.1 N NaOH, neutralized by washing with 5 x 100 ⁇ l of TE- L buffer, resuspended in 160 ⁇ l (Bam H I) or 280 ⁇ l (Sau3A I) of SB buffer and stored at 4°C.
  • FIG. 27 shows amplification of the primary PENTAmer libraries from human genomic DNA prepared by complete BamB I or partial Sau3Al digestion. Magnetic beads containing library DNA are prewashed in water and 0.5 ⁇ l of each library used as template for PCR amplification with 100 nM of universal adaptor A3 and adaptor Bl primers (primers 13 and 18) in final volume of 25 ⁇ l. After initial denaturing the indicated number of cycles are earned out at 94°C for 10 sec and 68°C for 75 sec. Ten ⁇ l aliquots are separated on 1 % agarose gel and visualized on Fluor S Multilmager (Bio Rad) after staining with Sybr Gold.
  • Fluor S Multilmager Bio Rad
  • This example describes the preparation of circular single-stranded derivatives of primary human genomic Sau3A I and BamB I libraries described in Example 10. These circular libraries are used as template for reverse PCR amplification with kernel human sequences keeping intact the adaptor tags which will allow simultaneous analysis of single nucleotide polymorphic (SNP) regions in multiple individuals.
  • SNP single nucleotide polymorphic
  • Magnetic beads containing primary human BamB I or Sau3A I library DNA are pre- washed in water and 0.5 ⁇ l of each library is used as template for PCR amplification in 16 individual tubes for each library with 200 nM of 5 '-biotinylated adaptor Bl primer (primer 19) and 5 '-phosphorylated adaptor A3 primer (primer 23) in final volume of 50 ml. After initial denaturing at 95°C, eighteen cycles of PCR are performed at 94°C for 10 sec and 68°C for 75 sec. Beads are removed on magnet and the individual PCR samples for each library are pooled.
  • Samples are purified free of primers and Taq polymerase on Qiaquick PCR purification filters (Qiagen; Valencia, CA) and eluted in 150 ⁇ l of 10 mM Tris-HCl, pH 8.5. DNA is polished with 4 units of T4 DNA Polymerase (Roche; Nutley, NJ) in the presence of 200 nM of each dNTP for 30 min at 25°C.
  • DNA samples are purified on Qiaquick PCR purification filters (Qiagen; Valencia, CA), supplemented with 1/4 volume of QF buffer (240 mM NaCl, 3% isopropanol, and 10 mM Tris-HCl, pH 8.5 final concentrations) in a volume of 400 ⁇ l, and centrifuged at 200 x g to a volume of 100 ⁇ l in Microcon YM-100 filtration units. The samples are washed 3 times with 400 ⁇ l of TE-L buffer at 200 x g and concentrated to a final volume of 130 ⁇ l.
  • QF buffer 240 mM NaCl, 3% isopropanol, and 10 mM Tris-HCl, pH 8.5 final concentrations
  • Sau3A I library DNA is incubated with 20 ⁇ l of 0.1 N NaOH for 5 min at room temperature. Eluted non-biotinylated DNA strands are neutralized with 10 ml of 0.2 N HCl and 3 ⁇ l of 1 M Tris-HCl, pH 8.0. Sample is diluted to 100 ⁇ l with water and any residual biotin-containing DNA is removed by incubation with 200 ⁇ g of fresh streptavidin beads for 30 min at room temperature. Single-stranded DNA is purified on Qiaquick PCR purification filters (Qiagen; Valencia, CA) and eluted in 60 ⁇ l of 10 mM Tris- HCl, pH 8.5.
  • Qiaquick PCR purification filters Qiagen; Valencia, CA
  • Sau3A I library single-stranded DNA is incubated with 3'-C7 amino blocked bridging oligonucleotide (primer 24) bringing together adaptor A3 (5' terminus) and adaptor Bl (3 '-terminus) to form circular molecules by ligation.
  • DNA is aliquoted into four 200 ng samples and incubated with bridging oligonucleotide (primer 24) at 0, 15, 75, or 150 nM final concentration in 1 x Tsc ligase buffer (Roche; Nutley, NJ) and final volume of 30 ⁇ l. After initial denaturing at 95°C for 1 min, ligation is performed for 24 cycles at 94°C for 20 sec and 65°C with 5 units of Tsc DNA ligase (Roche; Nutley, NJ).
  • Samples are split into two aliquots of 15 ⁇ l and one half is treated with 0.7 units of T4 DNA polymerase (Roche; Nutley, NJ) for 1 hr at 37°C in the absence of dNTPs to destroy linear DNA molecules. The remaining half is left untreated. Aliquots of each treated and untreated sample are analyzed on 6% TBE urea acrylamide gel (Novex; San Diego, CA) after staining with Sybr Gold (Molecular Probes; Eugene, OR). FIG. 28 shows the result of this analysis. In the samples receiving bridging oligonucleotide, a low mobility band appears conesponding to circularized PENTAmer molecules.
  • FIG. 29A shows that the amount of circular DNA molecules before treatment with the exonuclease activity of T4 polymerase is higher than the amount of circular and linear DNA after such treatment combined (compare lanes 2 and 4). This result independently validates the fo ⁇ nation of circular single-stranded library molecules.
  • FIG. 29 B shows an attempt for amplification of kernel human sequence in circular mode with a pair of primers specific for exon 10 of the human tp53 gene. The same template as in the experiment on FIG. 29 A but without dilution was used before or after treatment with exonuclease in 35 cycles of PCR amplification. The products of such amplification would be expected to have relatively uniform size distributed around the average length of termination of nick- translation of PENT molecules in the parental primary library. However, amplicons of multiple discrete lengths varying from 200 bp to 1 Kb are amplified, indicating more complex events compared to kernel amplification from linear library in nested mode (Example 12).
  • This example shows amplification of genomic kernel sequences from primary human BamBl and Sau3A I libraries by nested PCR.
  • first PCR reaction limited number of cycles are performed using the distal adaptor Bl primer (primer 18) and a kernel specific primer up to 500 bp downstream of BamB I or Sau3A I restriction sites.
  • second PCR is performed with the proximal adaptor Bl primer (primer 22) and nested kernel primers.
  • One microliter aliquots of the purified DNA samples from the first amplification are used as templates in second PCR with 50 nM proximal Bl adaptor primer (primer 22) and 200 nM nested kernel primer specific for exon 10 of the human tp53 gene which anneals 45 bp downstream of the kernel primer used in the first PCR amplification.
  • samples are subjected to 33 cycles at 94°C for 10 sec, and 68°C for 75 sec and 10 ⁇ l aliquots are analyzed on 1% agarose gel after staining with Sybr Gold (FIG. 30 A).
  • lane 1 is purified by Qiaquick PCR purification 'kit (Qiagen; Valencia, CA) and used as template for sequencing with both nested primers, 1 and 2 with DYEnamic ET terminator reagent mix (Amersham Pharmacia Biotech) and analyzed on MegaBACE 1000 capillary sequencing system (Amersham Pharmacia Biotech) as described in Example 6.
  • Additional sequences are amplified by PCR with adaptor Bl universal primers (primers 18 and 22) and the following pairs of nested primers: one specific for PENTAmer covering exons 2 and 3 of the human tp53 gene using BamBl library as template, and two covering exons 4 and 5, and 6, 7, and 8 respectively, using Sau3A I library as template (FIG. 31).
  • Primary and secondary (nested) PCR rounds are ca ⁇ ied out as described above.
  • the bands are excised from the agarose gel, extracted with Ultrafree DA gel extraction kit (Millipore; Bedford, MA) and appropriate dilutions are used as templates for re-amplification in individual PCR reactions with the same primers used in secondary PCR.
  • the amplification products are purified with Qiaquick PCR purification kit (Qiagen; Valencia, CA) and sequenced as above with the conesponding nested primers used in PCR.
  • kernel genomic sequences can be amplified after nested PCR from primary genomic human PENTAmer libraries prepared by complete or partial restriction digestion.
  • Vec-torette PCR a novel approach to genomic walking. PCR Methods Appl. i:39-42.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of prefe ⁇ ed embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

La présente invention concerne des procédés et des réactifs améliorés pour la marche sur le génome d'un acide nucléique. On génère une bibliothèque amplifiable de molécules de translation de coupure, et on procède à une marche sur le génome à partir d'une séquence connue dans l'acide nucléique par la production d'au moins une molécule de coupure de translation, le séquençage d'une partie de la molécule de coupure de translation, et la production d'une deuxième molécule de coupure de translation par l'amorce de la technique de la préamplification du génome à partir de la région de la séquence obtenue de la précédente molécule de coupure de translation.
PCT/US2001/044970 2001-05-02 2001-11-15 Marche sur le genome par l'amplification selective de bibliotheque d'adn de translation de coupure et l'amplification a partir de melanges complexes de matrices WO2002103054A1 (fr)

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WO2004081183A3 (fr) * 2003-03-07 2005-05-12 Rubicon Genomics Inc Immortalisation d'adn in vitro et amplification genomique complete a l'aide de bibliotheques generees a partir d'adn fragmente de manière aleatoire
US7670810B2 (en) 2003-06-20 2010-03-02 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
US7718403B2 (en) 2003-03-07 2010-05-18 Rubicon Genomics, Inc. Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
US7803550B2 (en) 2005-08-02 2010-09-28 Rubicon Genomics, Inc. Methods of producing nucleic acid molecules comprising stem loop oligonucleotides
US8206913B1 (en) 2003-03-07 2012-06-26 Rubicon Genomics, Inc. Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
WO2013036668A1 (fr) * 2011-09-06 2013-03-14 Gen-Probe Incorporated Gabarits mis sous forme circulaire pour séquençage
US8409804B2 (en) 2005-08-02 2013-04-02 Rubicon Genomics, Inc. Isolation of CpG islands by thermal segregation and enzymatic selection-amplification method
US8440404B2 (en) 2004-03-08 2013-05-14 Rubicon Genomics Methods and compositions for generating and amplifying DNA libraries for sensitive detection and analysis of DNA methylation
US9404147B2 (en) 2011-12-19 2016-08-02 Gen-Probe Incorporated Closed nucleic acid structures
CN109762919A (zh) * 2018-12-29 2019-05-17 浙江医药高等专科学校 一种快速鉴别覆盆子及其多种混淆品的方法
US10767208B2 (en) 2011-09-06 2020-09-08 Gen-Probe Incorporated Closed nucleic acid structures
WO2023196738A1 (fr) * 2022-04-05 2023-10-12 The Regents Of The University Of California Séquençage de nanopores d'arn à l'aide d'une transcription inverse

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Cited By (31)

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US7718403B2 (en) 2003-03-07 2010-05-18 Rubicon Genomics, Inc. Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
US11661628B2 (en) 2003-03-07 2023-05-30 Takara Bio Usa, Inc. Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
US8206913B1 (en) 2003-03-07 2012-06-26 Rubicon Genomics, Inc. Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
US11492663B2 (en) 2003-03-07 2022-11-08 Takara Bio Usa, Inc. Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
US10837049B2 (en) 2003-03-07 2020-11-17 Takara Bio Usa, Inc. Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
WO2004081183A3 (fr) * 2003-03-07 2005-05-12 Rubicon Genomics Inc Immortalisation d'adn in vitro et amplification genomique complete a l'aide de bibliotheques generees a partir d'adn fragmente de manière aleatoire
US9045796B2 (en) 2003-06-20 2015-06-02 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
US7670810B2 (en) 2003-06-20 2010-03-02 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
US11591641B2 (en) 2003-06-20 2023-02-28 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
US10738350B2 (en) 2003-06-20 2020-08-11 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
US8440404B2 (en) 2004-03-08 2013-05-14 Rubicon Genomics Methods and compositions for generating and amplifying DNA libraries for sensitive detection and analysis of DNA methylation
US9708652B2 (en) 2004-03-08 2017-07-18 Rubicon Genomics, Inc. Methods and compositions for generating and amplifying DNA libraries for sensitive detection and analysis of DNA methylation
US9598727B2 (en) 2005-08-02 2017-03-21 Rubicon Genomics, Inc. Methods for processing and amplifying nucleic acids
US8399199B2 (en) 2005-08-02 2013-03-19 Rubicon Genomics Use of stem-loop oligonucleotides in the preparation of nucleic acid molecules
US8778610B2 (en) 2005-08-02 2014-07-15 Rubicon Genomics, Inc. Methods for preparing amplifiable DNA molecules
US8728737B2 (en) 2005-08-02 2014-05-20 Rubicon Genomics, Inc. Attaching a stem-loop oligonucleotide to a double stranded DNA molecule
US7803550B2 (en) 2005-08-02 2010-09-28 Rubicon Genomics, Inc. Methods of producing nucleic acid molecules comprising stem loop oligonucleotides
US10196686B2 (en) 2005-08-02 2019-02-05 Takara Bio Usa, Inc. Kits including stem-loop oligonucleotides for use in preparing nucleic acid molecules
US10208337B2 (en) 2005-08-02 2019-02-19 Takara Bio Usa, Inc. Compositions including a double stranded nucleic acid molecule and a stem-loop oligonucleotide
US8071312B2 (en) 2005-08-02 2011-12-06 Rubicon Genomics, Inc. Methods for producing and using stem-loop oligonucleotides
US8409804B2 (en) 2005-08-02 2013-04-02 Rubicon Genomics, Inc. Isolation of CpG islands by thermal segregation and enzymatic selection-amplification method
US11072823B2 (en) 2005-08-02 2021-07-27 Takara Bio Usa, Inc. Compositions including a double stranded nucleic acid molecule and a stem-loop oligonucleotide
US10752944B2 (en) 2011-09-06 2020-08-25 Gen-Probe Incorporated Circularized templates for sequencing
US10767208B2 (en) 2011-09-06 2020-09-08 Gen-Probe Incorporated Closed nucleic acid structures
WO2013036668A1 (fr) * 2011-09-06 2013-03-14 Gen-Probe Incorporated Gabarits mis sous forme circulaire pour séquençage
US9404147B2 (en) 2011-12-19 2016-08-02 Gen-Probe Incorporated Closed nucleic acid structures
US11396673B2 (en) 2011-12-19 2022-07-26 Gen-Probe Incorporated Closed nucleic acid structures
US10184147B2 (en) 2011-12-19 2019-01-22 Gen-Probe Incorporated Closed nucleic acid structures
CN109762919B (zh) * 2018-12-29 2022-04-08 浙江医药高等专科学校 一种快速鉴别覆盆子及其多种混淆品的方法
CN109762919A (zh) * 2018-12-29 2019-05-17 浙江医药高等专科学校 一种快速鉴别覆盆子及其多种混淆品的方法
WO2023196738A1 (fr) * 2022-04-05 2023-10-12 The Regents Of The University Of California Séquençage de nanopores d'arn à l'aide d'une transcription inverse

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