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WO2009000281A1 - Procédés de clonage directionnel d'acides nucléiques amplifiés et leurs utilisations - Google Patents

Procédés de clonage directionnel d'acides nucléiques amplifiés et leurs utilisations Download PDF

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Publication number
WO2009000281A1
WO2009000281A1 PCT/EP2007/005514 EP2007005514W WO2009000281A1 WO 2009000281 A1 WO2009000281 A1 WO 2009000281A1 EP 2007005514 W EP2007005514 W EP 2007005514W WO 2009000281 A1 WO2009000281 A1 WO 2009000281A1
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Prior art keywords
nucleic acid
target nucleic
nicking
vector
enzyme
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PCT/EP2007/005514
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English (en)
Inventor
Fé Isabel GARCIA MACEIRA
Veónica Inmaculada LUNA GUERRERO
Gracia MONTERO PEÑALVO
Julio Manuel Martinez Moreno
Elier Paz Rojas
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Canvax Biotech, S.L.
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Priority to PCT/EP2007/005514 priority Critical patent/WO2009000281A1/fr
Publication of WO2009000281A1 publication Critical patent/WO2009000281A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Definitions

  • the present invention relates to the cloning of nucleic acids. More particularly, the invention relates to a simplified method for directional cloning of polymerase amplified nucleic acids after treatment with a nicking enzymes, into vectors with compatible ends.
  • nicking enzymes are endonucleases which cleave only a single strand of DNA duplex. Some nickases introduce single-stranded nicks only at particular sites on a DNA molecule by binding to and recognising a particular nucleotide recognition sequence.
  • the bases that result from nicking enzyme treatment at the end of amplified target nucleic acids have a low melting point so that at 37°C degrees they are released from the double strand.
  • the protruding ends generated could be cloned in a vector digested with any restriction endonu- clease that produces compatible ends with the nicking enzyme generated ends.
  • PCR has found numerous research applications, such as in the determination of genetic mutations, in engineering template-modified sequences using mismatched primer sequences, and in producing sufficient genetic material for direct sequencing. PCR has also been applied to many medical and legal problems where it has been used in such areas as diagnosis of monogenic diseases, analysis of biological evidence, etc. Further applications of PCR amplification are discussed in a number of references. See, e.g., PCR Technology, H. A. Erlich, ed., Stockman Press, 1989. Doubtless, many more applications will be forthcoming.
  • PCR may be satisfactory for certain applications often it is desirable to obtain a clone of PCR amplified products for further analysis, modification, or protein expression.
  • a number of mRNA species exhibit polymorphic transcripts.
  • Alternative splicing of the mRNA species to give multiple transcripts can be unambiguously sequenced after molecular cloning of the PCR amplification products (Frohman et al., Proc. Natl. Acad. Sci . USA 85: 8998-9002 (1988)). Cloning of PCR generated samples to construct cDNA libraries may also be desired.
  • TA cloning method The most common methods for cloning cDNA and PCR amplified fragments are the TA cloning method or the approaches involves incorporation of flanking restriction sites onto the ends of primer molecules. TA cloning method does not permit directional cloning of amplified fragments and although the incorporation of restriction sites allows this application, these method can result in unintended internal restriction of uncharacterized sequences .
  • Restriction enzymes are widely used in molecular biology. The continuously expanding panel of known restriction enzymes reveals them as major tools of recombinant DNA technology. Truth be told, many recombinant DNA technologies are unlikely to have been devel- oped without the discovery of restriction enzymes. The frequency with which a given restriction enzyme cuts DNA depends on the recognition site of the enzyme. With a quick calculation and a couple of basic assumptions, one can use this knowledge to estimate how frequently it should cut a piece of DNA. For example, an enzyme with four bases recognition sites (“four cutter”) should on average cut once every 256 base pairs, while a six cutter, once every 1028 base pairs. This fact gave molecular biologist the method they required to produce DNA fragments of known size for their experiments. Nevertheless, the presence of unwanted restriction enzyme recognition sites is a problem in some cases, for example, in the ORF cloning to expression in genome scale.
  • nickases are endonucleases capable of cleaving a particular strand of a DNA duplex, which strand may be determined by the orientation of the recognition sequence.
  • the nickases recognise an asymmetric recognition site, which means that in the recognition sequence one strand of the DNA duplex does not possess the same sequence as the complementary strand, when each strand is read in the 5' to 3' direction.
  • nicks can serve as initiation points for a variety of further enzymatic reactions such as replacement DNA synthesis, strand-displacement ampli- fication (Walker et al, Proc. Natl. Acad. Sci. USA 89: 392-396 (1992)), exonucleolytic degradation or the creation of small gaps (Wang et al, MoI. Biotechnol. 15: 97-104 (2000) ) .
  • LIC Ligation independent cloning
  • exonuclease III T4 or T7 DNA polymerases, Klenow polymerase, etc
  • Ligation independent cloning takes advantage of the 3' -> 5' exonuclease activity to create very specific 12- to 15- nucleotide single-stranded overhangs (Aslanidis et al, Nucleic Acid Res. 18: 6069- 6074 (1990); Haun et . al., Biotechniques 13: 515-518 (1992) ) .
  • the efficiency of the ligation independent procedure depends greatly on the length of nucleotide overhangs. Some authors refer that the highest effi- ciency of transformation is achieved with a length of 20 nucleotide and if this length is at least 5 nucleotides shorter or longer the efficiency of transformation with chimerical DNA molecules decreases a multiple of several times (Peacock S. L. et . al . Biochim. Biophys . Acta 655: 243-250 (1981)).
  • the annealed LIC vector and insert are transformed directly into E.coli competent cells, and covalent bonds are formed at the vector-insert junctions within the cell.
  • the ability of nickases to nick specifically pre-selected DNA strand is applied for efficient preparation of vectors for a ligation independent cloning procedure.
  • the cloning vector is designed with a unique restriction site flanked on each side by inverted recog- nition sequences for the nickase, such that the nickase is capable of cleaving different strands of the vector on each side of the recognition sequence.
  • the use of nicking enzymes for this application provides an alternative method of preparing the substrate for exonuclease degradation (US Patent 6.867.028).
  • nicking enzyme treatment to digest a target nucleic acid amplified with primers containing nicking enzyme recognition sequences to produce a protruding end of few bases.
  • the directional cloning using nicking enzymes is a novel approach that affords directional cloning even in the presence of additional nicking enzymes recognition sequences in target nucleic acids.
  • the method of invention satisfies the current demand for expressing multiple open reading frames in parallel and provides additional advantages in the state of art.
  • This invention discloses a method for directional cloning of a target nucleic acid to obtain recombinant nucleic acid molecules.
  • the method involves subjecting a target nucleic acid to PCR amplification with two primers designed so that at least one of them contain a nicking enzyme recognition site. Digestion of the target nucleic acid amplified with a nicking enzyme release one, or two, or three, or four, or five base(s) from the end of the double stranded DNA of target nucleic acid amplified to produce a protruding cohesive end with one, or two, or three, or four, or five base(s), respectively.
  • the amplified and digested target nucleic acid amplified is admixed with a vector digested with at least one restriction enzyme that generates compatible ends with the target nucleic acid ends .
  • the ligation reaction mixture combines the amplified and digested nucleic acid, the digested vector with compatible ends to the target nucleic acid ends and a prese- lected ligase enzyme.
  • the said admixture is maintained under predetermined reaction conditions for a sufficient period of time to effect ligation of the amplified and digested target nucleic acid and the digested vector to give the recombinant molecules.
  • the ligation mixture is then transformed in a suitable host cell line.
  • the primers are designed so that one primer contains a nicking enzyme recognition site that after nicking treatment generates a 3' or 5' over- hang.
  • the other primer used for amplification of target nucleic acid should contain a nicking enzyme recognition site.
  • the protruding ends generated by nickases need to be different sequences to afford directional cloning and should both 3' overhangs, both 5' overhangs, one 3' and the other 5' , one 3' overhang and the other a blunt end or one 5' overhang and the other a blunt end.
  • one primer should contain a nicking enzyme recognition site to generate after nickase action a 3' or a 5' overhang, while the other can be blunt ended by polymerase amplification, by restriction enzyme digestion or by nicking treatment.
  • the target nucleic acid should contain internal nicking enzyme recognition sites.
  • the cleave of those sequences away of the ends of target nucleic acid does not releases the polynucleotide generated by the nicking enzyme treatment.
  • the melting temperature of this polynucleotide is higher than that of a polynucleotide of a few bases generated at the end of target nucleic acid so that is not easy release them.
  • Those nicks, far of the end of target nucleic acid can be efficiently repaired by the T4-DNA ligase action during the ligation reaction.
  • the target nucleic acid is obtained from RNA retrotran- scribed to cDNA with a polythimidylate primer modified in the 5 ' end to include the recognition sequence of a nicking enzyme.
  • the other primer to amplification reaction should be designed with a nicking enzyme recognition site or not.
  • the tar- get nucleic acid could be one or more than one nucleic acid sequences so that the method should be applied in parallel cloning projects and in high throughput cloning projects .
  • the vector is digested with at least one restriction enzyme to generate two different non compatible ends in order to avoid recircularization of the vector.
  • the vector ends must be compatible with the target nucleic acid ends previously amplified and digested with nicking enzymes.
  • a vector of the present invention may be an expression vector, a cloning vector, a shuttle vector, and the like.
  • a vector contemplated by the present invention is at least capable of directing the replication, and preferably also expression, of a gene operatively linked to the vector.
  • a vector contemplated by the present invention includes a procaryotic repli- con, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombi- nant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a procaryotic repli- con i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombi- nant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • procaryotic replicons are well known in the art.
  • those embodiments that include a procaryotic replicon may also include a gene whose expression con- fers drug resistance to a bacterial host transformed therewith. Typical bacterial drug resistance genes are those that confer resistance to ampicillin, tetracycline, kanamycin or chloramphenicol.
  • Those vectors that include a procaryotic repli- con can also include a procaryotic promoter capable of directing the expression (transcription and translation) of the gene transformed therewith.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
  • Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a PCR product molecule of the present invention.
  • Expression vectors compatible with eucaryotic cells can also be used to form the recombinant DNA molecules of the present invention.
  • Eucaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired gene.
  • the eucaryotic cell expression vectors used to construct the recombinant DNA molecules of the present invention include a selection marker that is effective in a eucaryotic cell, preferably a drug resistance selection marker.
  • a preferred drug resistance marker is the gene whose expression results in antibiotic resistance, for example, the neomycin phosphotransferase (neo) gene (Southern et al . , J. MoI. Appl . Genet., 1:327-341 (1982) ) .
  • nicking enzymes are available commercially but not all of them are useful to apply in this method.
  • the preferred nicking enzymes are selected from those that have an asymmetric recogniton sites of 4, or 5, or 6, or 7, or 8 base pairs.
  • the preferred nicking enzymes are obtained by engineering of type HA restriction enzymes, that is engineering of restriction enzymes that have an asymmetric recognition sequence.
  • Further preferred nicking enzymes are those obtained from type HS, that is, restriction enzyme that cut outside their normally asymmetric recognition sequence .
  • the nicking enzymes are selected from a group of commercially available nicking enzymes that comprises, but it is not limited to, the enzymes Nt. AIwI or Nt.BstNBI or Nb. Btsl or Nb.BsrDI or Nb.Bsml.
  • nicking enzymes could be artificially engineered from restriction enzymes, selected from a group comprising, but not limited to the following enzymes: Aarl, AcIWI, Alw26I, Bbsl, Bbvl, BciVI, Bfil, Bful, BfuAI, Bmrl, Bpil(BbvII), Bpml , BpuEI, Bsal, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsmAI, BsmBI, BsmFI, BspMI, BspPI (Binl), Bsrl, BsrDI, BstF5I, Btsl, Bvel, Csel, Eamll04I (Ksp632I), Earl, Ecil, Eco31I, Eco57I, Eco57MI, Esp3I, Faql, Faul, Finl, Fokl, Gsul, Hgal,
  • kits suitable for directional cloning into a vector of amplified target nucleic acid having in at least one end a nicking recognition site are afforded by the present invention.
  • the kits include a vector having at least two restriction enzymes sequences in the multiple cloning site that generate non compatible ends or linearized plasmid with compatible ends to the target nucleic acid ends.
  • the kits also include a restriction enzyme capable of cleaving the vector and a nicking enzyme for cleaving target nucleic acids amplified with suitable primers.
  • the kits may include a ligase capable of ligating the heterologous strands of amplified target nucleic acid digested with a nickase and a linear vector having compatible ends.
  • Figure 1 presents the strategy to cloning GZH using nicking enzyme treatment of amplified fragment with pGZH-l_for and pGZH-2_rev.
  • GZH amplified fragment has an internal Nt.AIwI recognition on each DNA strand.
  • Figure 2 presents the strategy to cloning GZM using nicking enzyme treatment of amplified fragment with pGZM-l_for and pGZM-2_rev.
  • Figure 3 presents the strategy to cloning cDNA using nicking enzyme treatment of amplified fragment.
  • Figure 4 shows the strategy to cloning cDNA using olidT primer designed with a Ht. AIwI recognition site and a sense primer without nicking recognition sequence .
  • DNA molecules are said to have 5' ends and 3' ends because mononucleotides are joined to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the 5' end if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the 3' end if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
  • a double stranded nucleic acid molecule may also be said to have a 5' and 3' end, wherein the 5' refers to the end containing the accepted beginning of the particular region, gene or structure.
  • a nucleic acid sequence even if internal to a larger oligonucleotide, may also be said to have 5' and 3' ends that said fragment would have were it isolated from the larger oligonucleotide.
  • discrete elements may be referred to as being "upstream or 5' elements" and "downstream or 3' elements” .
  • Asymmetric Recognition Site Means that in the recognition sequence of the restriction enzyme or nicking enzyme, one strand of the DNA duplex does not possess the same sequence as the complementary strand, when each strand is read in the 5' to 3' direction.
  • Base pair(s) Abbreviation bp .
  • Base pair(s) Refers to the hydrogen bonded nucleotides of, for example, adenine (A) with thymidine (T) , or of cytosine (C) with guanine (G) in a double stranded DNA molecule. This term is also used generally as a unit of measure for DNA length.
  • Base pairs are said to be "complementary" when their component bases pair up normally by hydrogen bonding, such as when a DNA or RNA molecule adopts a double stranded configuration .
  • Compatible ends Ends are said to "compatible” if a) they are both blunt or contain complementary single strand extensions (such as that created after digestion with a restriction endonuclease) and b) at least one of the ends contains a 5' phosphate group. Compatible ends are therefore capable of being ligated by a double stranded DNA ligase (e.g. T4 DNA ligase) under standard conditions.
  • a double stranded DNA ligase e.g. T4 DNA ligase
  • Cloning or to clone Used in reference to an insert sequence and vector means ligation of the insert sequence into a vector capable of replicating in a host. These terms when used in reference to an insert sequence, a vector, and a host cell refers generally too making copies of a given insert sequence.
  • a piece of DNA e.g. insert sequence, amplified sequence
  • a vector e.g. plasmid
  • a host usually a bacterium
  • clone can also refer either to a bacterium carrying a cloned DNA, or to the cloned DNA itself.
  • Complementary Nucleotide Sequence A sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to another single strand to specifically (non-randomly) hybridize to it with consequent hydrogen bonding. This term is used to a sequence of nucleotides related by the base-pairing rules (A paired with T, C paired with G) .
  • Duplex DNA A double-stranded nucleic acid molecule comprising two strands of substantially complementary polynucleotides held together by one or more hydro- gen bonds between each of the complementary bases present in a base pair of the duplex.
  • duplex DNA refers to either a DNA-DNA duplex compris- ing two DNA strands (ds DNA) , or an RNA-DNA duplex comprising one DNA and one RNA strand.
  • Expression vector Recombinant DNA molecule containing a desired coding sequence and appropriate nu- cleic acid sequences necessary for expression of the operably linked coding sequence (e.g. insert sequence that codes for a product) in a particular host organism.
  • Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional) , and a ribosome binding site, often along with other sequences. While nucleic acid sequences necessary for expression in eucaryotes usually include a promoter, a Kozak sequence, a signal peptide and a terminator sequence.
  • Gene A nucleic acid whose nucleotide sequence codes for a RNA, DNA or polypeptide molecule. Genes may uninterrupted sequences of nucleotides or they may include such intervening segments as introns, promoter regions, splicing sites and repetitive sequences. A gene can be either RNA or DNA.
  • Host Cells refers to any cell that can be transformed with heterologous DNA (such as a vector) .
  • host cells include, but are not limited to, bacteria strains, yeast strains or mammalian cells.
  • nickase or nicking enzyme Two types of nicking enzymes are known. Herein refers to endonucleases which cleave only a single strand of a DNA duplex. Some nickases introduce single-stranded nicks only at particular sites on a DNA molecule, by binding to and recognising a nucleotide recognition sequence.
  • enzymes such as BpulOI where the wild type restriction enzyme has two subunits, each of which nicks a different strand, the mutant nicking enzymes made by inactivating one or the other subunit should be named Nt.
  • BpulOI for the enzyme that nicks the top strand of the normal recognition sequence and Nb.
  • BpulOI for the enzyme that nicks the bottom strand.
  • Nucleotide a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose) , a phosphate group, and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (I' carbon of the pentose) and that combination of base and sugar is a nucleoside.
  • the nucleoside contains a phosphate group bonded to the 3' or 5' position of the pentose it is referred to as a nucleotide.
  • a sequence of operatively linked nucleotides is typically referred to herein as a "base sequence” or “nucleotide sequence”, or “nucleic acid sequence” and their grammatical equivalents, and is represented herein by a formula whose left to right orientation is in the conventional direction of 5' terminus to 3' terminus.
  • Oligonucleotide Refers to a short length of single stranded polynucleotide chain. Oligonucleotides are typically less than 100 residues long. Oligonucleo- tides are often referred to by their length. For example a 6 residue oligonucleotide is referred to as a "6 mer”. Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides .
  • Polynucleotide a polymer of single or double stranded nucleotides.
  • polynucleotide and its grammatical equivalents will include the full range of nucleic acids .
  • a polynucleotide will typically refer to a nucleic acid molecule comprised of a linear strand of two or more deoxyribonucleotides and/or ribonucleotides. The exact size will depend on many factors, which in turn depends on the ultimate conditions of use, as is well known in the art.
  • the polynucleotides of the present invention include primers, RNA/DNA segments, oligonucleotides (relatively short polynucleotides), genes, vectors, plasmids, and the like.
  • Primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a template nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization, such as DNA polymerase, re- verse-transcriptase and the like, under suitable temperature and pH reaction conditions.
  • the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively double stranded. If double stranded, the primer is first treated to separate its strand before being used to prepare extension products .
  • Recombinant DNA molecule a DNA molecule pro- prised by operatively linking a nucleic acid sequence, such as a gene, to a DNA molecule sequence of the present invention.
  • a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in Nature.
  • Recombinant DNA's not having a common biological origin, i.e., evolutionarily different, are said to be "heterologous" .
  • Restriction site or enzyme recognition site Re ⁇ fers to a particular DNA sequence recognized by its cognate restriction endonuclease.
  • Type II restriction enzyme Refers to restriction enzymes that recognize specific DNA sequences and cleave at constant positions at or close to that sequence .
  • Transformation or transfection refers to the introduction of foreign DNA into cells. Transformation may be accomplished by a variety of means known to the art including, but not limiting to, calcium phosphate- DNA co-precipitation, DEAE-dextran mediated transfec- tion, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, viral infection, and biolistics.
  • Vector a DNA molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment.
  • a DNA segment e.g., gene or polynucleotide
  • vectors capable of directing the expression of genes encoding for one or more proteins are referred to herein as "expression vectors". Particularly important vectors allow cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase.
  • mM millimolar
  • pmol picomoles
  • g grams
  • mg milligrams
  • ug micrograms
  • ng nanograms
  • L liters
  • mL milliliters
  • uL microlliters
  • °C centigrade degrees
  • Luria Broth (LB) medium contained tryp- tone (10 g/L) , yeast extract (5 g/L) , sodium chloride (5 g/L) .
  • LB plates contained also agar (15 g/L) . LB plates were supplemented with ampicillin at 100 ug/mL before plating the media.
  • X-GaI plates contain LB agar supplemented with 0,005% X-GaI, 0,134 mM IPTG and ampicillin.
  • the EMBL Nucleotide Database Accession Number of pMBL plasmid is DQ059139. It has pUC replication origin, ampicillin resistance and the alpha peptide of lacZ driven by lac promoter.
  • the plasmid pGEX-4T-l was obtained from GE-
  • This plasmid is derived from pGEX-2T and contain a Tac promoter, the glutathione S-transferase
  • GST thrombin recognition site
  • pGEX-4T-l was modified in our lab to eliminate GST gene. The modified plasmid conserved the multiple cloning site of pGEX-4T-l.
  • Mini-prep plasmid DNA was purified by CTAB method
  • the present invention contemplates the amplification of target nucleic acids.
  • the PCR technique is described hereinbelow, to generate enhanced amounts of a target nucleic acid sequence.
  • Taq DNA polymerase reaction was performed in 20 uL. The reaction contained Taq polymerase buffer Ix, 15 pmol each primer, 0,8 mM dNTPs, 50 ng template plasmid DNA and IU of Taq DNA polymerase. PCR cycle conditions were 35 seconds at 94°C, 35 seconds at 60°C, 1 minute at 72°C for 25 cycles, followed by 7 minutes at 72°C.
  • Taq DNA polymerase or high fidelity polymerase available from a variety of sources .
  • the primers used herein are selected to be complementary to the different strands of each specific sequence to be synthesized or amplified.
  • the primer sequences may not reflect the exact sequence of the template because a non-complementary polynucleotide were attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand.
  • the primers are provided in single- stranded form for maximum efficiency.
  • the primers are synthetized by MWG using PSF technology to purify them.
  • Detection of amplified nucleic acid product can be accomplished by any of a variety of well known techniques.
  • the amplified product is separated on the basis of molecular weight by gel electrophoresis, and the separated products are then visualized by the use of nucleic acid specific stains which allow one to observe the discrete species of resolved amplified product present in the gel.
  • nucleic acid specific stains which allow one to observe the discrete species of resolved amplified product present in the gel.
  • numerous nucleic acid specific stains exist and would be suitable to visualize the electrophoretically separated nucleic acids, ethidium bromide is preferred.
  • Ligation Reactions The ligation reactions were performed in 10 uL adding T4 DNA ligase buffer IX, 10 ng of vector, insert in molar relation with vector 5:1 and 5 U Weiss of T4 DNA ligase. Ligation reactions were incubated at 22°C during at least 1 hour. Any ligase available commercially is contemplated to perform the ligation reaction effectively using methods and conditions well known to those skilled in the art. A pre- ferred ligase is T4 DNA ligase.
  • Transformation Procedure Frozen chemically competent E.coli cells were thawed in ice. To 50 uL of cells added 1-5 uL of ligation mix. This mixture was incubated in ice during 5 minutes and spreaded onto X- GaI plates with ampicillin preincubated at 37°C. The above protocol is routinely used in our lab. The plates were incubated overnight at 37°C.
  • the transformation can be done with other chemi- cal protocols or by electroporation using electrocompe- tent cells.
  • Competence determination The efficiency of chemically competent cells was determined by transformation with supercoiled plasmid pUC19. Typically, 10 pg of pUC19 was mixed with the competent cells, transformed and spread onto LB plates with ampicillin. The plates were incubated overnight at 37°C. The number of colonies was counted to calculate the efficiency in colony forming units (CFU) /ug pUC19.
  • CFU colony forming units
  • the efficiency of competent cells used in the following examples ranged from 10 sup. 7 (10 7 ) to 10 sup. 8 (10 8 ) CFU/ug pUC19.
  • the presence of recombinant DNA can be detected by well known immunological methods when the recombinant DNA is capable of directing the expression of a subject polypeptide.
  • cells successfully transformed with a subject recombinant DNA containing an expression vector produce a polypeptide displaying a characteristic antigenicity.
  • Samples of a culture containing cells suspected of being transformed are harvested and assayed for a subject polypeptide using antibodies specific for that polypeptide antigen, such as those produced by an appropriate hybridoma .
  • the present method entails a culture comprising a nutrient medium containing host cells transformed with a recombinant DNA molecule of the present invention that is capable of expressing a gene encoding a subject polypeptide.
  • the culture is maintained for a time period sufficient for the transformed cells to express the subject polypeptide.
  • the expressed polypeptide is then recovered from the culture.
  • compositions and Kits The compositions serving as host vectors can be packaged in kit form.
  • packaging refers to a solid matrix or material customarily utilized in a system and capable of holding within fixed limits one or more of the reagent components for use in a method of the present invention.
  • materials include glass and plastic (e.g., polyeth- ylene, polypropylene and polycarbonate) bottles, vials, paper, plastic and plastic-foil laminated envelopes and the like.
  • a package can be a glass vial used to contain the appropriate quantities of polynucleotide primer(s), or plasmids, or nicking and restriction enzyme (s), or DNA polymerase, or DNA ligase, or a combination thereof. An aliquot of each component sufficient to perform at least one program of cloning will be provided in each container.
  • the reagent species, indicating means or primer extension reaction reagents of any system described herein can be provided in solution, as a liquid dispersion or as a substantially dry powder, e.g., the plas- mids may be provided in lyophilized form.
  • the reagent species or indicating means is an enzyme
  • the enzyme's substrate can also be provided in a separate package of a system.
  • a solid support and one or more buffers can also be included as separately packaged elements in this system.
  • Example 1 Directional cloning of an amplified fragment that contain an internal nickase recognition site in each strand.
  • This example describes the cloning of an amplified fragment that contain internal inverted nickase recognition sites.
  • the fragment was amplified from the plasmid LIFESEQ 1879670 (Open Biosystems) using the flanking primers pGZH-l_for (5' GAT CCT AAG ATC CTG AGA ACA TGC AGC CAT TCC T 3', SEQ ID No.l) and pGZH-2 rev (5' TCG ATA TCG ATC CTT AGA GGC GCT TCA TTG TTC TC 3', SEQ ID No.2) .
  • AIwI recognition sequence are underlined in both primer. Nt. AIwI recognize the sequence 5'GGATCNNNNi3' 3 'CCTAGNNNN 5' and cleave only on the top strand. Note that the recognition site is asymmetric therefore one strand of the DNA duplex does not possess the same sequence as the complementary strand, when each strand is read in the 5' to 3' direction.
  • the nickase treatment of amplified fragment gen- erate one 5' overhang end complementary to BgIII restriction enzyme terminus and one 5' overhang end complementary to Sail ended.
  • GZH amplified fragment contains an internal Nt. AIwI recognition site in each strand .
  • Figure 1 shows the strategy to obtain GZH fragment flanked by BgIII and Sail compatible ends using nicking enzyme treatment of amplified fragment.
  • GZH fragment was amplified with the primers pGZH-l_for and pGZH-2_rev.
  • the primers and the sequences that they introduce are in grey, nicking enzyme recognition sites are inserted in a box.
  • the amplification reaction was loaded on a 0,7% agarose gel.
  • the band of 780 bp was purified and treated with the nicking enzyme Nt. AIwI (New England Biolabs).
  • the nicking reaction was incubated at 37°C during 1 hour .
  • the pMBL plasmids was separately treated with Bg111, EcoRl and Sail restriction enzymes using reaction conditions recommended by the enzyme supplier, MBI Fermentas.
  • EcoRl is located between BgIlI and Sail in pMBL multicloning site. The digestion with EcoRl reduces the background of vector without insert.
  • the linearized plasmid was separated from the small fragments released between the Bg111 and Sail sites by electrophoresis on a 0,7% agarose gel and the linearized plasmids were removed from the gel as described in Experimental Section.
  • the purified linearized plasmids and the amplified fragment treated with nickase were mixed in the ratio of 1:5, and ligated with T4 DNA ligase under reaction conditions described by the supplier.
  • the reaction products formed were used to transform cells for assay of cloning efficiency as described herein.
  • the plasmid without insert had a blue phenotype while the plasmid with insert, a white phenotype.
  • 81,6% of clones were recombinants.
  • This example illustrate the utility of the invention in a sequence that contain internal nickase recognition sites in each strand, overlapped in 6 base pairs.
  • Example 2. Directional cloning of an amplified fragment with two internal nickase recognition sites .
  • This example describes the cloning of an ampli- fied fragment with two internal nickase recognition sites.
  • the amplified fragment of GZM contain two internal Nt. AIwI recognition sites, one at aproximately 100 base pairs of 5' end and the other, at aproximately 50 base pairs of 3' end.
  • the fragment was amplified from the plasmid LIFESEQ 1875481 (Open Biosystems) using the flanking primers pGZM-l_for (5' GAT CCT AAG ATC CGG CCC GAT GGA GGC TTG CGT G 3', SEQ ID No .3 ) and pGZM-2_rev (5' TCG ATA TCG ATC CCA TCA CCC CAG AGC ATC AGG C 3', SEQ ID No .4 ) .
  • Nt. AIwI recognition site are underlined in both primers.
  • the nickase treatment generate one 5' overhang end complementary to BgIII restriction enzyme terminus and the other, 5' overhang end complementary to Sail ended.
  • Figure 2 shows the strategy to obtain GZM fragment flanked with BgIII and Sail compatible ends using nicking enzyme treatment of amplified fragment.
  • the primers used in the amplification reaction were pGZM- l_for and pGZM-2_rev.
  • the primers and the sequences that they introduce are in grey, nicking enzyme recognition sites are inserted in a box.
  • the amplification reaction was loaded on a 0,7% agarose gel.
  • the band of 820 bp was purified and treated with the nicking enzyme Nt. AIwI (New England Biolabs).
  • the nicking reaction was incubated at 37°C during 1 hour .
  • the pMBL plasmids was separately treated with BgIII, EcoRI and Sail restriction enzymes using reaction conditions recommended by the enzyme supplier, MBI Fermentas. EcoRI is located between BgI11 and Sail in pMBL multicloning site. The digestion with EcoRI reduces the background of vector without insert.
  • the linearized plasmid was separated from the small fragments released between the BgIII and Sail sites by electrophoresis on a 0,7% agarose gel and the linearized plasmids were removed from the gel as described in Experimental Section.
  • the purified linearized plasmids and the amplified fragment treated with nickase were mixed in the ratio of 1:5, and ligated with T4 DNA ligase under reaction conditions described by the supplier.
  • the reaction products formed were used to transform cells for assay of cloning efficiency as described herein with the results presented in Table 2.
  • the plasmid without insert had a blue phenotype while the plasmid with insert, white phenotype.
  • Recombi- nant clones represents 91,33% of total clones.
  • Example 3 Directional cloning of an amplified cDNA fragment with both primers containing nickase recognition site.
  • This example describes the directional cloning of an amplified cDNA fragment using both primers with nickase recognition site.
  • the fragment was amplified from total RNA by RT-PCR, using the flanking primers olidT-Nt.AlwI (5'TCG ATA TCG ATC CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T 3', SEQ ID No.5) and pGZH-l_for (5' GAT CCT AAG ATC CTG AGA ACA TGC AGC CAT TCC T 3', SEQ ID No .1 ) .
  • the olidT primer is designed with Nt. AIwI recognition site so that the nickase treatment generates one 5' overhang end complementary to Sail restriction enzyme terminus and the other, is an specific sense primer containing Nt. AIwI recogniton site so that the nickase treatment generates another 5' overhang, in this case complementary to BgIII restriction enzyme.
  • olidT primer was used as antisense primer in reverse transcription and in PCR reaction were used both olidT-Nt . AIwI and the specific sense primer.
  • Figure 3 shows the strategy to cloning cDNA using nicking enzyme treatment of amplified fragment.
  • the primers and the sequences that they introduce are in grey, nicking enzyme recognition sites are inserted in a box.
  • the amplification reaction was loaded on a 0,7% agarose gel and the band was purified and treated with the nicking enzyme Nt. AIvI (New England Biolabs). The nicking reaction was incubated at 37°C during 1 hour.
  • the pGEX-4T-l plasmid (GE Healthcare) without the glutathione S-transferase (GST) was separately treated with Bamtil and Sail restriction enzymes using reaction conditions recommended by the enzyme supplier, MBI Fermentas.
  • the linearized plasmid was separated from the small fragments released between the Bamtil and Sail sites by electrophoresis on a 0,7% agarose gel and the linearized plasmids were removed from the gel as described in Experimental Section.
  • the purified linearized plasmid and the ampli- fied fragment treated with nickase were mixed in the ratio of 1:5, and ligated with T4 DNA ligase under reaction conditions described by the supplier.
  • the reaction products formed were used to transform cells for assaying the cloning efficiency as described herein- below with the results presented in Table 3.
  • the plasmid without insert had a blue phenotype while the plasmid with insert, a white phenotype.
  • Recombinant clones represents 88,27% of total clones. This example illustrates the efficiency of this method to generate protruding ends in an amplified cDNA fragment.
  • Example 4 Directional cloning of an amplified cDNA fragment with only olidT primer with nickase recognition site.
  • This example describes the directional cloning of an amplified cDNA fragment using only one of both primer with nickase recognition site.
  • the fragment was amplified from total RNA by RT-PCR, using the flanking primers olidT-Nt .
  • AIwI (5'TCG ATA TCG ATC CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T 3', SEQ ID No.5) and pGZM-3_for (5' ATG GAG GCC TGC GTG TCT TCA 3', SEQ ID No.6) .
  • the olidT primer is designed with nickase Nt. AIwI recognition site so that the nickase treatment generate one 5' overhang end complementary to Sail restriction enzyme terminus and the other, is a specific sense primer without nicking or restriction enzyme recognition sites.
  • the olidT primer was used as an- tisense primer in reverse transcription and in PCR reaction were used both olidT-Nt . AIwI and the specific sense primer.
  • the enzyme used in PCR reaction was a high fidelity DNA polymerase, Pfu DNA polymerase.
  • Figure 4 shows the strategy to directional cloning of cDNA amplified with olidT with nicking enzyme recognition site and a primer without nicking enzyme recognition site.
  • the primers and the sequences that they introduce are in grey, nicking enzyme recognition site are inserted in a box.
  • the amplification reaction was loaded on a 0,7% agarose gel and the amplified band was purified and treated with the nicking enzyme Nt.
  • AIwI New England
  • the pGEX-4T-l plasmid without GST was separately treated with Smal and Sail restriction enzymes using reaction conditions recommended by the enzyme supplier, MBI Fermentas .
  • the linearized plasmid was separated from the small fragments released between the Smal and Sail sites by electrophoresis on a 0,7% agarose gel and the linearized plasmids were removed from the gel as described in Experimental Section.
  • the purified linearized plasmids and the ampli- fied fragment treated with nickase were mixed in the ratio of 1:5, and ligated with T4 DNA ligase under reaction conditions described by the supplier.
  • the reaction products formed were used to transform cells for assaying the cloning efficiency as described with the results presented in Table 4, where the plasmid without insert had a blue phenotype while the plasmid with insert, a white phenotype.
  • This example shows that the method of invention can be applied when only one primer contain a nicking enzyme recognition site so that the nickase treatment generate a protruding end while the other has a blunt end.
  • Recombinant clones represents 98,87% of total clones .

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Abstract

L'invention concerne des procédés de production de molécules d'ADN recombinant à partir de vecteurs hôtes appropriés digérés avec des endonucléases de restriction et des acides nucléiques cibles soumis à une amplification avec des amorces comprenant des séquences de reconnaissance pour enzymes de coupure et la digestion par des enzymes de coupure qui produisent des extrémités saillantes dans l'acide nucléique cible. Ce procédé tire avantage du point de fusion bas de la base ou des bases résultant du traitement avec les enzymes de coupure à l'extrémité des acides nucléiques cibles amplifiés. Cette base ou ces bases, une fois libérées de l'extrémité bicaténaire, produisent une extrémité compatible avec un vecteur digéré avec une endonucléase de restriction sélectionnée de manière appropriée, lequel peut être ligaturé et transformé dans un hôte compatible pour obtenir une molécule d'ADN recombinant cloné de manière réactionnelle.
PCT/EP2007/005514 2007-06-22 2007-06-22 Procédés de clonage directionnel d'acides nucléiques amplifiés et leurs utilisations WO2009000281A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107012205A (zh) * 2010-08-17 2017-08-04 凯杰有限公司 使用切口酶的解旋酶依赖性等温扩增

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WO2001094544A2 (fr) * 2000-06-02 2001-12-13 New England Biolabs, Inc. Clonage et production de l'endonuclease de coupure n.$m(f)i$m(g)bst$m(f)/i$m(g)nbi et procedes associes d'utilisation des endonucleases de coupure dans l'amplification par deplacement de simple brin
EP1176204A1 (fr) * 2000-07-24 2002-01-30 Fermentas AB Nucléase
WO2002059357A2 (fr) * 2001-01-24 2002-08-01 Genomic Expression Aps Dosage et kit d'analyse d'expression genique
EP1264881A2 (fr) * 2001-06-01 2002-12-11 New England Biolabs, Inc. Production par génie génétique de endonucleases de coupure simple-brin à partir de endonucleases de restriction de type IIs
US20030022317A1 (en) * 2000-12-15 2003-01-30 New England Biolabs, Inc. Use of site-specific nicking endonucleases to create single-stranded regions and applications thereof
WO2003087301A2 (fr) * 2002-04-12 2003-10-23 New England Biolabs, Inc. Procedes et compositions destines a la manipulation de l'adn
WO2007135354A1 (fr) * 2006-05-19 2007-11-29 Plant Bioscience Limited Clonage moléculaire amélioré à base d'excision de l'uracile

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Publication number Priority date Publication date Assignee Title
WO2001094544A2 (fr) * 2000-06-02 2001-12-13 New England Biolabs, Inc. Clonage et production de l'endonuclease de coupure n.$m(f)i$m(g)bst$m(f)/i$m(g)nbi et procedes associes d'utilisation des endonucleases de coupure dans l'amplification par deplacement de simple brin
EP1176204A1 (fr) * 2000-07-24 2002-01-30 Fermentas AB Nucléase
US20030022317A1 (en) * 2000-12-15 2003-01-30 New England Biolabs, Inc. Use of site-specific nicking endonucleases to create single-stranded regions and applications thereof
WO2002059357A2 (fr) * 2001-01-24 2002-08-01 Genomic Expression Aps Dosage et kit d'analyse d'expression genique
EP1264881A2 (fr) * 2001-06-01 2002-12-11 New England Biolabs, Inc. Production par génie génétique de endonucleases de coupure simple-brin à partir de endonucleases de restriction de type IIs
WO2003087301A2 (fr) * 2002-04-12 2003-10-23 New England Biolabs, Inc. Procedes et compositions destines a la manipulation de l'adn
WO2007135354A1 (fr) * 2006-05-19 2007-11-29 Plant Bioscience Limited Clonage moléculaire amélioré à base d'excision de l'uracile

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107012205A (zh) * 2010-08-17 2017-08-04 凯杰有限公司 使用切口酶的解旋酶依赖性等温扩增

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