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WO1991004340A1 - Amplification isothermique in vitro de l'acide nucleique - Google Patents

Amplification isothermique in vitro de l'acide nucleique Download PDF

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Publication number
WO1991004340A1
WO1991004340A1 PCT/US1990/005321 US9005321W WO9104340A1 WO 1991004340 A1 WO1991004340 A1 WO 1991004340A1 US 9005321 W US9005321 W US 9005321W WO 9104340 A1 WO9104340 A1 WO 9104340A1
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WIPO (PCT)
Prior art keywords
dna
interest
sequence
region
promoter
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PCT/US1990/005321
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English (en)
Inventor
Jeffrey C. Olson
Kary B. Mullis
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Cambridge Biotech Corporation
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Publication of WO1991004340A1 publication Critical patent/WO1991004340A1/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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Definitions

  • PCR polymerase chain reaction
  • the method is designed to enable the rapid and specific in vitro amplification of target nucleic acid sequence using the reiterative, reciprocal interactions of two synthetic primer oligonucleotides with their respective templates and an inducing agent of template-directed synthesis.
  • PCR and the transcription-based amplification method are time-consuming and labor-intensive
  • the PCR method is tedious because thermal cycling, by hand or by machine, is necessary.
  • the transcription-based amplification method requires that additional reagents be added periodically due to the inactivation of necessary enzymes by the temperatures employed.
  • a cost effective, isothermal method of amplifying a specific nucleic acid sequence would be of great benefit.
  • the present invention pertains to an isothermal method for amplifying DNA of interest which has a defined 3' end or RNA of interest.
  • the DNA of interest which has a defined 3' end or RNA of interest.
  • RNA of interest can be present in a mixture of sequences or purified and can be a portion of a longer sequence, referred to as target DNA and target RNA, respectively.
  • target DNA and target RNA can be processed by a target DNA sequence in which a DNA sequence to be amplified (DNA of interest) is present, to produce DNA of interest having a defined 3' end available for hybridization with complementary nucleic acid sequences and subsequently amplifying the DNA of interest.
  • a target DNA sequence containing DNA of interest which has a defined 3' end is combined with at least two selected primers and a number of selected enzymes, to produce an overall cyclic, isothermal reaction which results in amplification of the DNA of interest.
  • the present invention provides a "one-pot", isothermal amplification method for amplifying DNA of interest which has a defined 3' end and is contained in a target DNA sequence.
  • the specific amplification of DNA of interest can be accomplished at a wide variety of temperatures (i.e., any temperature consistent with the stability of the required enzymes and integrity of the various nucleic acid sequences involved).
  • This is accomplished by employing a number of selected enzymes including, for example, ribonuclease H (RNase H), which functions to degrade RNA hybridized to a DNA sequence.
  • RNase H ribonuclease H
  • the present invention provides an isothermal amplification method which avoids the time-consuming, heating and cooling manipulations necessary to denature
  • the present invention provides a "one-pot" amplification reaction whereby a single mixture of amplification reagents is combined with a target DNA sequence in which the DNA of
  • the method described herein can be used to amplify and subsequently detect and/or characterize DNA of interest associated with various pathogens, infectious diseases, and genetic disorders.
  • the amplification method can be utilized to clone DNA of interest for insertion into a suitable expression vector.
  • Figure 1 is a schematic representation of one embodiment of the method of the present invention, in which a restriction enzyme which recognizes and specifically cleaves single-stranded DNA at a selected site is employed.
  • Figur-e 2 is a schematic representation of another embodiment of the method of the present invention, in which a modified oligonucleotide having a region comprising a compound which specifically cleaves single-stranded DNA at a selected site is employed.
  • Figure 3 is a schematic representation of an embodiment of the method of the present invention, in which a DNA oligonucleotide and a restriction enzyme which recognizes and specifically cleaves
  • double-stranded DNA at a selected site are employed.
  • Figure 4 is a schematic representation of an embodiment of the method of the present invention, in which a restriction oligonucleotide, a restriction complement oligonucleotide and a restriction enzyme which recognizes double-stranded DNA and specifically cleaves outside its recognition site at a selected site are employed.
  • Figure 5 is a schematic representation of an embodiment of the method of the present invention, in which a promoter primer complement oligonucleotide is employed.
  • Figure 6 is a schematic representation of an embodiment of the method of the present invention, in which a restriction oligonucleotide and a restriction enzyme which recognizes single-stranded DNA and specifically cleaves outside its recongition site at a selected site are employed.
  • Figure 7 is a schematic representation of an embodiment of the method of the present invention, in which RNA of interest is amplified.
  • Figure 8 is a schematic representation of an embodiment of the method of the present invention, in which a DNA-dependent RNA polymerase which recongizes single-stranded DNA and nonspecifically snythesizes RNA is employed.
  • Figure 9 is a schematic representaion of the method of the present invention whereby a region of the ampicillin resistance gene is amplified.
  • the present invention pertains to a method for the amplification of RNA or DNA.
  • the quantity of selected DNA or RNA (referred to, respectively, as DNA of interest and RNA of interest) is increased as desired by combining the necessary reagents and maintaining the resulting combination under appropriate conditions.
  • the present method is particularly useful because it is not a multi-step procedure (i.e., does not require
  • the DNA or RNA of interest to be amplified can be only a portion of a longer sequence, referred to, respectively, as a target DNA or target RNA sequence, or can constitute the entire target DNA or target RNA sequence (in which case, the DNA of interest or RNA of interest and the target DNA or target RNA sequence, respectively, are the same).
  • the target DNA or RNA sequence can be obtained from various sources, such as from cloned DNA, from plasmids or obtained from various naturally-occuring sources, including
  • bacteria bacteria, yeast, viruses, and higher organisms such as plants or animals, in purified or non-purified form.
  • DNA or RNA to be amplified which is DNA of interest having a defined 3' end or RNA of interest, is combined with the following reagents: two
  • RNA-dependent DNA polymerase a DNA-dependent DNA polymerase
  • DNA-dependent DNA polymerase a DNA-dependent DNA polymerase
  • DNA-dependent RNA polymerase an enzyme or an agent with an RNase H activity; (i.e., an enzyme or an agent which has the ability to degrade the RNA strand of a RNA/DNA heteroduplex); and appropriate nucleoside triphosphates.
  • the two DNA primers used are referred to as a promoter primer and a reverse primer.
  • the promoter primer is an oligonucleotide which includes at least two regions whose presence is necessary for the promoter primer to be useful in the present method: a 5' region which includes a recognition sequence for a DNA-dependent RNA polymerase and a 3' region which includes a sequence which is complementary to a region of the DNA of interest or the RNA of interest.
  • DNA sequences may be inserted into the sequence of the promoter primer between the 5' end containing the DNA-dependent RNA polymerase promoter sequence and the 3' end which is complementary to the DNA or RNA of interest. This makes it possible to include
  • DNA or RNA of interest which would not normally be found there.
  • a DNA fragment will be produced which has both the target DNA sequence, the DNA-dependent RNA polymerase
  • the reverse primer is an oligonucleotide whose sequence is homologous to a region of the DNA of interest or the RNA of interest which is 5' to and nonoverlapping with the region of the DNA of interest or RNA of interest to which the promoter primer is complementary.
  • the reverse primer may also have additional selected sequence at its 5', end which will be incorporated into the resultant DNA fragment during the amplification process.
  • the reverse primer is capable of hybridizing to a RNA strand or a DNA strand which is complementary to the RNA of interest or the DNA of interest.
  • DNA of Interest must be processed in order for it to become a substrate for amplification.
  • One method of processing is to make a complementary RNA copy of the DNA of interest, which is able to react directly in the amplification
  • DNA of interest which has a defined 3' end is defined as DNA of interest which terminates at its 3' end in a sequence which is sufficiently
  • the subject method requires reagents which act as a RNA-dependent DNA polymerase; a DNA-dependent DNA polymerase; a DNA-dependent RNA polymerase; and an enzyme or an agent with RNase H activity.
  • reagents which act as a RNA-dependent DNA polymerase; a DNA-dependent DNA polymerase; a DNA-dependent RNA polymerase; and an enzyme or an agent with RNase H activity.
  • Each of these four functions can be carried out by a separate reagent; in this case, four
  • the enzyme reverse transcriptase can function as a RNA-dependent DNA polymerase and as a DNA-dependent DNA polymerase and can also exhibit an RNase H-like activity.
  • nucleoside triphosphates which include the necessary DNA and RNA precursors for amplification of target DNA are also included in the reaction mixture in adequate amounts. These include the deoxyribonucleoside 5'-triphosphates dATP, dCTP, dGTP and TTP and the ribonucleoside triphosphates ATP, CTP, GTP, and UTP. In addition, a molar excess of the oligonucleotide primers (as compared to target RNA or DNA present) is added to the buffer containing the target DNA or RNA.
  • the necessary reagents and the target DNA or target RNA are combined, they are maintained under conditions (e.g., time, temperature) appropriate for the reagents to function and amplification to occur.
  • the method proceeds as follows: the 3' end of the promoter primer hydridizes to the defined 3' end of the DNA of interest. Through the action of a DNA-dependent DNA polymerase, the promoter primer is extended in the 3' direction, resulting in production of a sequence complementary to the DNA of interest, thus making the DNA of interest double-stranded.
  • the DNA of interest is also extended from its 3' end, resulting in a sequence complementary to the RNA promoter sequence and, thus, making the RNA promoter region of the promoter primer
  • the double-stranded promoter-DNA of interest product is used as a template by the double-stranded promoter-DNA of interest product.
  • RNA molecules which have a defined 5' end and are complementary to the DNA of interest.
  • the reverse primer hybridizes to the RNA strand at its site of complementarity (i.e., to a nonoverlapping region 3' of the region on the RNA strand to which the promoter primer is homologous).
  • the reverse primer is extended by an RNA-dependent DNA polymerase.
  • An agent with RNase H-like activity is an agent with RNase H-like activity.
  • RNA/DNA heteroduplex degrades the RNA strand from the newly formed RNA/DNA heteroduplex, producing a
  • the defined 3' end of the DNA fragment is able to hybridize to the 3' end of the promoter primer.
  • the 3' end of the promoter primer is extended by a DNA-dependent DNA polymerase, which produces a complementary copy of the DNA fragment making that region double-stranded.
  • the 3' end of the DNA fragment is extended through the activity of the DNA-dependent DNA polymerase and, as a result, the RNA promoter region is made double-stranded.
  • RNA molecules having a defined 5' and 3' end which are complementary to the target "DNA of interest".
  • RNA molecules having a defined 5' and a defined 3' end can be amplified using the promoter primer, reverse primer and reagents, as described above.
  • Amplification of RNA of interest by the present method proceeds as follows: The 3' end of the
  • selected promoter primer hybridizes to its
  • RNA-dependent DNA polymerase By the action of a RNA-dependent DNA polymerase, the promoter primer is extended from its 3' end. This results in production of DNA complementary in sequence to that of the RNA of interest.
  • An agent with an RNase H An agent with an RNase H
  • the reverse primer hybridizes to a region 3' to and nonoverlapping with the promoter primer sequence contained at the 5' defined end of this single-stranded DNA and is extended in the 3' direction through the action of a DNA-dependent DNA polymerase, to produce a double-stranded DNA molecule which includes the RNA promoter sequence and the double- stranded DNA copy of the target RNA.
  • the double-stranded DNA molecule which includes the RNA promoter sequence is used by a DNA-dependent RNA polymerase to produce RNA molecules with a defined 5' end which is complementary to the original target RNA strand.
  • the reverse primer hybridizes to the RNA strand at its site of complementarity, 3' to and nonoverlapping with the region of promoter primer homology.
  • the reverse primer is extended by a RNA dependent-DNA polymerase.
  • An agent with RNase H-like activity e.g., which hydrolyzes RNA from an RNA/DNA heteroduplex degrades the RNA strand from the newly formed RNA/DNA heteroduplex. This step leaves a single-stranded DNA sequence with a defined 3' end and a defined 5' end.
  • the defined 3' end of the DNA sequence is able to hybridize to the 3' end of the promoter primer and the 3' end of the promoter primer is extended by a DNA-dependent DNA polymerase,
  • RNA sequence DNA-dependent RNA polymerase, which produces a RNA sequence with a defined 5' and 3' end.
  • This RNA sequence can be amplified using the promoter primer and reverse primer as described above.
  • a target DNA sequence in which DNA of interest is available for hybridization is combined with the reagents necessary for amplification to be carried out.
  • the reagents used in each embodiment are described below.
  • a restriction enzyme which recognizes and cleaves single-stranded DNA at a selected site is employed (see Figure 1).
  • restriction enzyme is combined, under appropriate conditions, with a target DNA sequence; a promoter primer; a reverse primer; at least one RNA-dependent DNA polymerase; at least one DNA-dependent DNA
  • RNA polymerase at least one DNA-dependent RNA polymerase; an agent with RNase H activity; and appropriate nucleoside triphosphates.
  • amplification of the DNA of interest occurs spontaneously.
  • the target DNA sequence is cleaved at a selected site by the restriction enzyme, which recognizes and specifically cleaves
  • DNA-dependent DNA polymerase using the DNA of interest as a template, and the 3' end of the DNA of interest is extended by the DNA-dependent DNA polymerase, using the promoter primer as a template, to produce a double-stranded DNA sequence having a double-stranded promoter.
  • this double-stranded DNA sequence is transcribed by the DNA-dependent RNA polymerase to produce a RNA transcript having a defined 5' end.
  • the reverse primer hybridizes to a complementary 3' region of the RNA transcript and the 3' end of the reverse primer is extended by the RNA-dependent DNA polymerase, using the RNA transcipt as a template.
  • the product of this extension process is a heteroduplex molecule comprising the RNA transcript having a defined 5' end and a reverse primer extension product having a sequence which corresponds to the DNA of interest .
  • the RNA transcript having a defined 5' end and hybridized to the reverse primer extension product is hydrolyzed by the agent with RNase H activity.
  • a 3' region of the promoter primer hybridizes to a complementary 3' region of the reverse primer extension product.
  • the 3' end of the promoter primer is extended by the DNA-dependent DNA
  • RNA transcript having a defined 5' end, a defined 3' end and a sequence complementary to the DNA of interest is used as a template in additional amplification cycles, thereby resulting in amplification of the DNA of interest.
  • a modified oligonucleotide is used to produce DNA of interest having a defined 3' end available for
  • the modified oligonucleotide is complementary to a region within the target DNA sequence adjacent to the defined 3' end of the DNA of interest.
  • the 3' end of the olignucleotide is
  • the modified oligonucleotide hybridizes to the complementary region of the target DNA sequence and the compound is activated, allowing it to specifically cleave the single-stranded DNA target DNA sequence at the selected site, to produce DNA of interest having a defined 3' end available for hybridization.
  • This DNA of interest having a defined 3' end is amplified using the primers and enzymes described above, in an isothermal, cyclic amplification reaction.
  • restriction enzyme are used to produce DNA of interest having a defined 3' end available for hybridization.
  • the oligonucleotide is complementary to a region of DNA of interest comprising a sequence which corresponds to the recognition site of a restriction enzyme which is capable of recognizing and specifically cleaving double-stranded DNA at a selected site adjacent to the defined 3' end of the DNA of interest.
  • a restriction enzyme which is capable of recognizing and specifically cleaving double-stranded DNA at a selected site adjacent to the defined 3' end of the DNA of interest.
  • the oligonucleotide hybridizes to the complementary region of the target DNA sequence and the enzyme which specifically cleaves double-stranded DNA cleaves the target DNA sequence at a selected site, to produce DNA of interest having a defined 3' end available for hybridization.
  • This DNA of interest having a defined 3' end is amplified using a promoter primer, a reverse primer and the enzymes described above, according to the amplification method of the present invention.
  • FIG. 4 Another embodiment of the method of the present invention, represented in Figure 4, utilizes a
  • restriction oligonucleotide a restriction complement oligonucleotide and a restriction enzyme to produce DNA of interest having a defined 3' end available for hybridization.
  • the restriction oligonucleotide has a 3' region complementary to a region within the target DNA sequence adjacent to the defined 3' end of the DNA of interest and a 5' region comprising a sequence which corresponds to the recognition site of a
  • restriction enzyme which recognizes double-stranded DNA and specifically cleaves outside Its recognition site.
  • the restriction enzyme which recognizes double-stranded DNA and specifically cleaves outside Its recognition site.
  • oligonucleotide is combined with a restriction
  • oligonucleotide hybridizes to the 5' region of the restriction primer to form a restriction complex having a double-stranded recognition site for the enzyme which recognizes double-stranded DNA and specifically cleaves outside its recognition site.
  • the restriction complex has a region complementary to a region within the target DNA sequence adjacent to the defined 3' end of the DNA of interest.
  • the restriction complex hybridizes to the target DNA sequence and the restriction enzyme which recognizes double-stranded DNA and specifically cleaves outside its recognition site cleaves the target DNA sequence at a selected site, to produce DNA of interest having a defined 3' end available for hybridization.
  • this DNA of interest is combined with a promoter primer, a reverse primer and the selected enzymes described above, to amplify the DNA of
  • a promoter-complement primer which has a region complementary to the promoter sequence of the promoter primer described above, is used in the amplification method, to amplify the DNA of interest contained in target DNA.
  • promoter-complement primer is combined with the target DNA sequence, the promoter primer, the reverse primer, and the selected enzymes described above.
  • promoter-complement primer hybridizes to the promoter sequence of the promoter primer, to produce a
  • double-stranded promoter complex hybridizes to the 3' end of the DNA of interest.
  • the 3' end of the promoter primer is extended by the DNA-dependent DNA polymerase using the DNA of interest as a template, to produce a
  • double-stranded DNA sequence having a double-stranded promoter.
  • This double-stranded DNA sequence is an intermediate in the amplification process described above, which proceeds to result in amplification of the DNA of interest.
  • a restriction primer is used to produce DNA of interest having a defined 3' end available for hybridization.
  • the restriction primer has a 3' region complementary to a region within the target DNA sequence adjacent to the defined 3' end of the DNA of interest and a 5' region comprising a sequence which corresponds to the recognition site of a restriction enzyme which recognizes single-stranded DNA and specifically cleaves outside its recognition site at a selected site.
  • the restriction primer is combined with the target DNA sequence, a promoter primer, a reverse primer, and the enzymes described above to amplify the DNA of interest.
  • the 3' region of the restriction primer hybridizes to the complementary region of the target DNA sequence.
  • the target DNA sequence is cleaved at a selected site by the
  • restriction enzyme which recognizes single- stranded DNA and specifically cleaves outside its recognition site, to produce DNA of interest having a defined 3' end available for hybridization. This DNA of interest having a defined 3' end is amplified in the
  • the target DNA sequence is combined with a DNA-dependent RNA polymerase which recognizes single-stranded DNA and nonspecifically synthesizes RNA using the DNA of interest as a template.
  • a RNA/DNA heteroduplex molecule is produced by the
  • RNA sequence is produced by the DNA-dependent RNA
  • RNA sequence synthesized using the DNA of interest as a template is combined with two primers; a promoter primer having a 5' region comprising a DNA-dependent RNA polymerase promoter sequence and a region 3' of the promoter sequence complementary to a 3' region of the RNA sequence synthesized using the DNA of interest as a template; and a reverse primer having a region complementary to the 3' end of the DNA of interest.
  • a promoter primer having a 5' region comprising a DNA-dependent RNA polymerase promoter sequence and a region 3' of the promoter sequence complementary to a 3' region of the RNA sequence synthesized using the DNA of interest as a template
  • a reverse primer having a region complementary to the 3' end of the DNA of interest.
  • the above-described enzymes and appropriate nucleoside triphosphates are included in the reaction mixture, to result in amplification of the DNA of interest
  • DNA of interest refers to DNA contained within a target DNA sequence, which can be either a single-stranded or double-stranded nucleic acid, to be amplified in the method of the present invention.
  • the DNA of interest has both a
  • oligonucleotide primers can be provided which are sufficiently complementary to their
  • the defined 3' end of the DNA of interest is that region of the DNA of interest to which a 3' region of a promoter primer is complementary and hybridizes thereto.
  • the defined 3' end is defined by cleavage of the target DNA sequence, to produce DNA of interest having a defined 3' end available for hybridization of the promoter primer and subsequent extension of both the promoter primer and the 3' end of the DNA of interest, to produce a double-stranded DNA sequence having a double-stranded promoter.
  • the DNA of interest is contained within a target DNA sequence which can be a single-stranded or a double-stranded nucleic acid sequence.
  • the DNA of interest can be only a portion of the target DNA sequence.
  • the DNA of interest can be the entire target DNA sequence, such that the terms "DNA of interest” and "target DNA sequence” refer to the same discrete molecule.
  • the target DNA sequence is treated, if necessary, to render the DNA of interest available for hybridization.
  • the strands are separated (e.g., by heat denaturation or enzyme separation) to produce two separate strands.
  • only a portion of the double-stranded target DNA sequence is separated to render the DNA of interest available for hybridization. This can be accomplished by a variety of known enzymes.
  • oligonucleotide as used in the method herein in referring to primers, probes, or fragments thereof, is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides.
  • the length of the oligonucleotide depends on many factors, which in turn depends on the ultimate function or use of the oligonucleotide.
  • primer refers to an oligonucleotide, which occurs naturally or is produced synthetically, and capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced.
  • an inducing agent such as a DNA-dependent DNA polymerase or a RNA-dependent DNA polymerase, and at a suitable temperature and pH
  • the primer acts as a point of initiation of synthesis.
  • the primer can be of various lengths, but must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. Primers useful in the method of the present invention are described in detail in U.S. Patent No. 4,683,202, the contents of which are incorporated herein by reference.
  • amplification method for processing the target DNA sequence to produce DNA of interest having a defined 3' end and subsequently amplifying the DNA of interest having a defined 3' end.
  • At least two primers are necessary for the amplification process: a "promoter primer” and a “reverse primer”, each of which is described above.
  • the primers used are "sufficiently" complementary to their respective target nucleic acid sequence to be amplified.
  • the promoter primer is sufficiently complementary to the defined 3' end of the DNA of interest contained in a target DNA
  • the primers must be sufficiently complementary in nucleic acid sequence to the target nucleic acid sequence for hybridization to occur.
  • primer sequence need not be the exact complement of the template.
  • a primer sequence need not be the exact complement of the template.
  • non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand.
  • the promoter primer has a 5' region comprising a DNA-dependent RNA polymerase promoter sequence.
  • non-complementary sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the nucleic acid sequence of the DNA of interest that hybridization occurs.
  • the oligonucleotide primers can be produced using any one of various methods, such as, for example, the phosphotriester and phosphodiester methods or automated embodiments thereof. Methods for synthesizing oligonucleotides are described in detail in U.S.
  • Patent No. 4,683,202 the contents of which have been previously incorporated herein. It is also possible to use a primer which has been isolated from a
  • the reverse primer is selected having a region complementary to a 3' region of a RNA transcript synthesized by a
  • RNA transcript is complementary to the DNA of interest.
  • the reverse primer has a region which "corresponds to”, or is substantially homologous to a 5' region of the DNA of interest. The degree of homology depends on many factors, including the degree of complementarity of the reverse primer for the RNA transcript.
  • restriction enzyme refers to an enzyme capable of specifically cleaving either single-stranded or double-stranded DNA at a selected site (I.e., at or near a specific nucleotide sequence).
  • a restriction enzyme which recognizes and specifically cleaves single-stranded DNA at a selected site is used in the
  • enzymes which can be used to cleave single-stranded DNA are selected from, but not limited to: Hha 1, BstN 1, Dde 1, Hae 3, Hga 1, Hinf 1, Hinp 1, Mnl 1, Rsa 1, Taq 1.
  • an enzyme which recognizes and specifically cleaves double-stranded DNA at a selected site can be used to produce DNA of interest having a defined 3' end.
  • enzymes which can be used to cleave double-stranded DNA are selected from, but not limited to: EcoR1, BamH1, Stu1, Hindlll, Seal, Haelll.
  • Fok 1 is an enzyme which specifically cleaves outside its recognition site.
  • Fok 1 is an enzyme which
  • Mnl 1 is an enzyme which catalyzes the hydrolysis of a protein which catalyzes the hydrolysis of a protein having the same function as a protein having the same function as a protein having the same function as a protein having the same function as a protein having the same function as a protein having the same function as a protein having the same function as a protein having the same function as a protein having the same function as a protein having the same function as ase, Szybalski, W., Gene, 40:175-182 (1985).
  • Mnl 1 is an enzyme which catas the hydrolysis of a protein having the same.
  • a non-enzymatic agent or compound which cleaves single-stranded DNA can be attached to a DNA probe to specifically cleave the target DNA sequence at a selected site, to produce DNA of
  • ethylenediaminetetracetic acid or diethylenediaminepentacetic acid can be covalently attached to a DNA probe and, in the
  • An inducing agent, or agent which catalyzes the primer extension reaction can be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes.
  • DNA-dependent DNA polymerase and RNA-dependent DNA polymerase.
  • Suitable enzymes for this purpose include, but are not limited to, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, reverse transcriptase, other enzymes, including heat-stable enzymes, which will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each nucleic acid strand.
  • reverse transcriptase can be used
  • RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase.
  • the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the appropriate template (e.g., the DNA of interest) and at the 3' end of the appropriate template and proceed in the 5' direction along the primer, until synthesis terminates in both directions.
  • Inducing agents which initiate synthesis at the 5' end and proceed in the 3' direction can also be used, as described above for those which initiate synthesis at the 3' end.
  • RNA-dependent RNA polymerase is necessary for the isothermal amplification method described herein.
  • the DNA-dependent RNA polymerase recognizes a double- stranded RNA polymerase promoter sequence and synthesizes a RNA transcript using the appropriate DNA sequence as a template.
  • Some examples of useful RNA polymerases are: T3, T7, SP6.
  • a T7 RNA polymerase is used which
  • RNA transcript recognizes single-stranded DNA and nonspecifically synthesizes a RNA transcript.
  • the T7 RNA polymerase synthesizes a RNA sequence using a single-stranded DNA sequence as a template, to produce a heteroduplex molecule (Chamberlin, M. and Ring, J., Journal ofBiological Chemistry, 248:2235-2244 (1973)).
  • RNA transcript is combined, under appropriate conditions, with the target DNA sequence; a promoter primer; a reverse primer; selected enzymes; and nucleoside triphosphates in the one-pot,
  • the T7 RNA polymerase can first be combined with a target DNA sequence under conditions appropriate for the synthesis of a RNA transcript, using the target DNA sequence as a template, to produce a heteroduplex molecule.
  • This heteroduplex molecule can be separated by heat denaturation or enzyme separation.
  • An agent with an RNase H-like activity which hydrolyzes RNA present in a RNA/DNA heteroduplex can be any compound which will function to separate a RNA sequence hybridized to a DNA sequence.
  • RNase H ribonuclease H specifically degrades RNA which is hybridized to DNA.
  • RNase H avoids the time-consuming task of separate heating and cooling manipulations necessary to denature RNA/DNA heteroduplexes and provides for an isothermal amplification reaction.
  • the amplification of the DNA of interest occurs under appropriate conditions in a one-pot, isothermal, cyclic, reaction employing a number of selected enzymes and at least two primers.
  • the appropriate conditions for the amplification process to occur include a buffered aqueous solution,
  • a target DNA sequence containing DNA of interest is combined, under appropriate conditions, with at least two primers, several selected enzymes and appropriate nucleoside triphosphates, to amplify the DNA of interest having a defined 3' end.
  • amplification reagents are combined, amplification of the DNA of interest occurs spontaneously.
  • the target DNA sequence treated, if necessary, to render the DNA of interest available for hybridization, is combined with the following: a promoter primer; a reverse primer; at least one RNA-dependent DNA
  • the promoter primer is selected having a 5' region comprising a DNA-dependent RNA polymerase promoter sequence and a region 3' of the promoter sequence complementary to the defined 3' end of the DNA of interest. When the 3' region of the promoter primer hybridizes to the 3' end of the DNA of
  • the 5' region comprising a promoter sequence remains single-stranded.
  • the present embodiment employs a restriction enzyme which recognizes and specifically cleaves single-stranded DNA at a selected site.
  • a restriction enzyme which specifically cleaves the target DNA sequence at a selected site (i.e., at or near the defined 3' end of the DNA of interest)
  • the restriction enzyme first cleaves the target DNA sequence at a selected site, to produce DNA of interest having a defined 3' end available for hybridization.
  • the 3' region of the promoter primer hybridizes to the complementary defined 3' end of the DNA of interest. Subsequently, the 3' end of the promoter primer is extended by a DNA-dependent DNA polymerase using the DNA of interest as a template. In addition, the 3' end of the DNA of interest is extended by the
  • the result of this extension process is a double-stranded DNA sequence having a
  • the double-stranded DNA comprises the DNA of interest extension product, the promoter primer extension product and a
  • DNA-dependent RNA polymerase recognizes the
  • RNA double-stranded promoter region of the double-stranded DNA and transcribes the DNA, to produce a RNA
  • RNA transcript having a defined 5' end.
  • the RNA transcript has a sequence which is complementary to the DNA of interest and has a defined 5' end complementary to the defined 3' end of the DNA of interest (i.e., the 5' end of the RNA transcript is defined by the 3' end of the DNA of interest produced by enzymatic cleavage of the target DNA sequence).
  • a reverse primer is necessary for amplification of the DNA of interest.
  • the reverse primer has a region complementary to a 3' region of a RNA transcript synthesized by a DNA-dependent RNA polymerase using the DNA of interest as a template. This RNA
  • the reverse primer hybridizes to the complementary 3' region of the RNA transcript having a defined 5' end.
  • the 3' end of the reverse primer is extended by a RNA-dependent DNA polymerase using the RNA transcript as a template, to produce a heteroduplex molecule comprising the RNA transcript and a reverse primer extension product having a sequence which is homologous to the DNA of interest.
  • the reverse primer extension product has a nucleic acid sequence substantially homologous to the DNA of Interest. The degree of homology depends on several factors, including the degree of complementarity of the reverse primer for the RNA transcript.
  • the RNA transcript hybridized to the reverse primer extension product is hydrolyzed by an agent with RNase H activity, which recognizes a RNA sequence hybridized to a DNA sequence and degrades the RNA sequence.
  • the reverse-primer extension product is available for hybridization with complementary nucleic acids.
  • the reverse primer extension product has a sequence which corresponds to the DNA of interest.
  • promoter primer which has a 3' region complementary to the 3' end of the DNA of interest is also
  • the promoter primer hybridizes to the complementary 3' region of the reverse-primer extension product. Subsequently, the 3' end of the promoter primer is extended by the DNA-dependent DNA polymerase using the reverse primer extension product as a template and the 3' end of the reverse primer extension product is extended by the DNA-dependent DNA polymerase using the promoter- containing primer as a template.
  • double-stranded DNA sequence having a defined 3' end, a defined 5' end and a double-stranded promoter. This DNA sequence having a double stranded promoter
  • the double-stranded DNA comprises a sequence which corresponds to the DNA of interest (i.e., the reverse primer extension product which is substantially homologous to the DNA of interest) and a sequence which is complementary to the DNA of interest (i.e., the promoter primer extension product).
  • the 3' end and the 5' end of the double-stranded DNA are defined. What this means is that the 3' end and the 5' end are available for hybridization (i.e., the 3' end and 5' end at a particular nucleic acid).
  • the nucleic acid sequence at the 3' end and the 5' end of the reverse primer extension product corresponds to the sequence at the 3' end and the 5' end of the DNA of interest and the sequence at the 3' end and the 5' end of the promoter primer extension product is complementary to the 3' end and the 5' end of the DNA of interest.
  • the double-stranded DNA sequence has a double-stranded DNA-dependent RNA polymerase promoter.
  • the double-stranded DNA sequence having a defined 3' end, a defined 5' end and a double-stranded
  • RNA transcript having a defined 5' end, a defined 3' end and a sequence complementary to the DNA of interest.
  • RNA transcript has a defined 3' end and a defined 5' end complementary to the nucleic acid sequences at the 3' end and the 5' end of the DNA of interest. This RNA transcript is used as a template in additional
  • a modified oligonucleotide is employed.
  • the modified oligonucleotide is employed.
  • oligonucleotide is selected to be complementary to a region within a target DNA sequence adjacent to the defined 3' end of the DNA of interest with a 3' end comprising a compound which specifically cleaves single-stranded DNA.
  • the modified oligonucleotide hybridizes to the target DNA sequence at a location 3' of the defined 3' end of the DNA of interest.
  • the 3' end of the modified oligonucleotide hybridizes to the target DNA sequence, the 3' end of the modified
  • oligonucleotide which comprises the compound which specifically cleaves single-stranded DNA is brought in proximity with the 3' defined end of the target DNA to be cleaved.
  • the modified oligonucleotide is combined, under appropriate conditions, with the target DNA sequence; a promoter primer; a reverse primer; the above-described enzymes; and appropriate nucleoside triphosphates.
  • the modified oligonucleotides hybridizes to the complementary region of the target DNA sequence (i.e., a region adjacent to the defined 3' end of the DNA of
  • the target DNA sequence is cleaved at a selected site by the compound which specifically cleaves single-stranded DNA, to produce DNA of interest having a defined 3' end.
  • the DNA of interest having a defined 3' end is amplified as described above in Section I. III. Use of a restriction enzyme which recognizes and specifically cleaves double-stranded DNA at a selected site.
  • a DNA oligonucleotide is used in combination with a restriction enzyme which recognizes and specifically cleaves double-stranded DNA at a selected site.
  • the DNA oligonucleotide is selected having a 5' region complementary to a region within the target DNA sequence adjacent to the defined 3' end of the DNA of interest and a 3' region comprising a sequence which corresponds to the recognition site of the restriction enzyme which recognizes and specifically cleaves double-stranded DNA at a selected site.
  • the 5' region of the DNA oligonucleotide can hybridize to the target DNA sequence at a location 3' of the defined 3' end of the DNA of interest.
  • the DNA oligonucleotide When hybridized, the DNA oligonucleotide forms a double-stranded complex which the appropriate restriction enzyme recognizes; the target DNA sequence is cleaved by the restriction enzyme to produce DNA of interest having a defined 3' end available for hybridization.
  • the DNA oligonucleotide is combined, under appropriate conditions, with the target DNA sequence; the appropriate restriction enzyme which recognizes and specifically cleaves double-stranded DNA at a selected site; a promoter primer; a reverse primer; the enzymes described above; and appropriate nucleoside triphosphates.
  • the DNA nucleotide hybridizes to the complementary region of the target DNA sequence.
  • the target DNA sequence is cleaved at a selected site by the enzyme which specifically cleaves double- stranded DNA, to produce DNA of interest having a defined 3' end.
  • the DNA of interest having a defined 3' end cleaved by the restriction enzyme is amplified using the method described above in Section I.
  • two additional oligonucleotides are used to produce DNA of interest having a defined 3' end available for hybridization.
  • a restriction oligonucleotide is selected having a 3' region complementary to a region within the target DNA sequence adjacent to the defined 3' end of the DNA of interest and a 5' region comprising a sequence which corresponds to the recognition site of a restriction enzyme which recognizes double-stranded DNA and specifically cleaves outside its recognition site.
  • restriction oligonucleotide hybridizes to the target
  • a second oligonucleotide referred to as a restriction complement oligonucleotide, which has a region complementary to the 5' region of the restriction oligonucleotide, hybridizes to the 5' region of the restriction oligonucleotide.
  • the restriction oligonucleotide and the restriction complement primer form a double- stranded complex comprising the recognition site of the restriction enzyme which recognizes double- stranded DNA and specifically cleaves outside its recognition site.
  • the restriction oligonucleotide and the restriction complement oligonucleotide are combined, under appropriate conditions, with the target DNA sequence; the appropriate restriction enzyme which recognizes double-stranded DNA and specifically cleaves outside its recognition site; a promoter primer; a reverse primer; the above-described enzymes; and appropriate nucleoside triphosphates.
  • the restriction complement oligonucleotide hybridizes to the 5' region of the restriction oligonucleotide to form a restriction complex having a double-stranded recognition site for the enzyme which recognizes double-stranded DNA and specifically cleaves outside its recognition site.
  • the restriction complex also has a region complementary to a region within the target DNA sequence adjacent to the defined 3' end of the DNA of interest.
  • the restriction complex hybridizes to the target DNA sequence. Subsequently, the target DNA sequence is cleaved at a selected site by the restriction enzyme which recognizes double-stranded DNA and specifically cleaves outside its recognition site, to produce DNA of interest having a defined 3' end.
  • This DNA of interest is amplified according to the method described in Section I above.
  • a promoter primer complement oligonucleotide is selected having a region complementary to the promoter sequence of the promoter primer. This oligonucleotide is combined, under appropriate
  • the promoter primer complement oligonucleotide hybridizes to the promoter sequence of the promoter primer, to produce a double-stranded promoter complex.
  • the double-stranded promoter complex hybridizes to the defined 3' end of the DNA of
  • the 3' end of the promoter primer is extended by a DNA-dependent DNA polymerase using the DNA of interest as a template, to produce a
  • double- stranded DNA sequence having a double-stranded promoter A DNA-dependent RNA polymerase recognizes the double-stranded promoter region and transcribes the double-stranded DNA sequence, to produce a RNA transcript having a defined 5' end.
  • RNA transcript having a defined 5' end hybridizes to the RNA
  • the reverse primer extension product has a sequence which corresponds to the DNA of interest.
  • the RNA transcript hybridized to the reverse primer extension product is hydrolyzed by an agent with RNase H activity. The hydrolysis of the RNA transcript leaves the reverse primer extension product available for hybridization.
  • the reverse primer extension product has a sequence which
  • the 3' region of the promoter primer hybridizes to a
  • the 3' end of the promoter primer is extended by a DNA-dependent DNA polymerase using the reverse primer extension product as a template and the 3' end of the reverse primer extension product is extended by the DNA-dependent DNA polymerase using the promoter primer as a template.
  • the result of this extension process is a double-stranded DNA sequence having a defined 5' end, a defined 3' end and a double-stranded promoter. This double-stranded DNA sequence is transcribed by a DNA-dependent RNA
  • RNA transcript having a defined 5' end, a defined 3' end and a sequence complementary to the DNA of interest. This RNA transcript is used as a template in additional
  • a restriction oligonucleotide is employed in combination with a restriction enzyme which recognizes
  • the restriction oligonucleotide is selected having a 3' region complementary to a region within the target DNA sequence adjacent to the defined 3' end of the DNA of interest and a 5' region comprising a sequence which corresponds to the recognition site of the restriction enzyme which recognizes single-stranded DNA and specifically cleaves outside its recognition site at a selected site.
  • the 3' region of the restriction oligonucleotide can be selected having a 3' region complementary to a region within the target DNA sequence adjacent to the defined 3' end of the DNA of interest and a 5' region comprising a sequence which corresponds to the recognition site of the restriction enzyme which recognizes single-stranded DNA and specifically cleaves outside its recognition site at a selected site.
  • the restriction oligonucleotide forms a complex which the restriction enzyme recognizes and cleaves outside its recognition site to cleave the target DNA sequence at a selected site.
  • the target DNA sequence is combined, under appropriate conditions, with the restriction oligonucleotide; the appropriate
  • restriction enzyme which recognizes single-stranded DNA and specifically cleaves outside its recognition site at a selected site; a promoter primer; a reverse primer; the above-described enzymes; and appropriate nucleoside triphosphates.
  • the 3' region of the restriction oligonucleotide hybridizes to the
  • the target DNA sequence is cleaved at a selected site by the restriction enzyme which
  • the DNA of interest having a defined 3' end is subsequently amplified according to the method described above in Section I.
  • the target DNA sequence is combined, under appropriate conditions, with a DNA-dependent RNA polymerase which recognizes single-stranded DNA and nonspecifically synthesizes RNA, using the DNA of interest as a template.
  • a DNA-dependent RNA polymerase which recognizes single-stranded DNA and nonspecifically synthesizes RNA, using the DNA of interest as a template.
  • the result is that a RNA/DNA heteroduplex molecule is produced.
  • single-stranded RNA is produced by the DNA-dependent RNA polymerase, using the RNA/DNA heteroduplex as a template.
  • RNA transcript synthesized using the DNA of interest as a template is combined with two selected primers, the enzymes described above, and appropriate
  • One of the primers is a promoter primer which has a 5' region comprising a DNA-dependent RNA polymerase promoter sequence and a region 3' of the promoter sequence complementary to a 3' region of the RNA sequence synthesized using the DNA of interest as a template.
  • the second primer is a reverse primer which has a region complementary to the 3' end of the DNA of interest.
  • the promoter primer hybridizes to the complementary 3' region of the RNA sequence synthesized by the
  • RNA sequence is hydrolyzed by an agent which catalyzes the hydrolysis of RNA present in a RNA/DNA
  • the promoter primer extension product After hydrolysis of the RNA transcript, the promoter primer extension product is available for hybridization with complementary nucleic acids.
  • the promoter primer extension product has a sequence which corresponds to the DNA of interest.
  • the reverse primer which has a 3' region complementary to the 3' end of the DNA of interest is also complementary to the 3' end of the promoter primer extension product.
  • the 3' region of the reverse primer hybridizes to the complementary 3' region of the promoter primer
  • the 3' end of the reverse primer is extended by a DNA-dependent DNA polymerase, using the promoter primer extension product as a template, to produce a double-stranded DNA sequence having a defined 5' end, a defined 3' end and a double-stranded promoter.
  • This double-stranded DNA sequence is transcribed by a DNA-dependent RNA polymerase, to produce a RNA transcript having a defined 5' end, a defined 3' end and a sequence which corresponds to the DNA of interest.
  • This RNA transcript is used as a template in additional amplification cycles, thereby resulting in amplification of the DNA of interest.
  • the present Invention pertains to a one-pot, isothermal method for amplifying DNA of interest which has a defined 3' end and is contained in a target DNA sequence.
  • the amplification method of the present invention results in an increase in the amount of DNA of interest or RNA of interest.
  • the present invention can be used for improving the efficiency of cloning DNA of interest contained in a target DNA sequence and for amplifying DNA of interest contained in a target DNA sequence to facilitate detection and/or identification.
  • the present method herein can be used to enable detection and/or characterization of DNA of interest associated with pathogens, infectious diseases, genetic disorders and cellular disorders.
  • Amplification is useful when the amount of DNA of interest available for analysis is very small, as, for example, in the prenatal diagnosis of sickle cell anemia using DNA obtained from fetal cells.
  • Genetic diseases can include specific deletions and/or
  • genomic DNA from an organism For example, sickle cell anemia, cystic fibrosis,
  • ⁇ -thalassemia ⁇ -thalassemia, ⁇ -thalassemia, and the like. These genetic diseases can be detected by amplifying the appropriate DNA of interest and analyzing it by
  • infectious diseases can be diagnosed by the presence in clinical samples of DNA of interest characteristic of the pathogen. These include
  • bacteria such as Salmonella, Chlamydia, and
  • viruses such as the hepatitis viruses
  • protozoan parasites such as the Plasmodium
  • the method herein can be utilized to clone DNA of interest for insertion into a suitable expression vector.
  • the vector can then be used to transform an appropriate host organism to produce the gene product of the DNA of interest by standard methods of
  • a small sample of DNA of interest can be amplified to a convenient level and then a further cycle of extension reactions performed wherein nucleotide derivatives which are readily detectable (such as 32 P-labeled or biotin labeled nucleoside triphosphates) are incorporated directly into the final DNA product, which can be analyzed by restriction and electrophoretic separation or any other appropriate method.
  • nucleotide derivatives which are readily detectable such as 32 P-labeled or biotin labeled nucleoside triphosphates
  • amplification process i.e., the promoter primer and reverse primer
  • the promoter primer and reverse primer need not be exactly complementary to the DNA of interest which is being amplified. It is only necessary that they be sufficiently complementary to hybridize to their respective targets to be
  • the product of an amplification reaction wherein the primers employed are not exactly complementary to the original template will contain the sequence of the primer rather than the template, thereby introducing an in vitro mutation. In further cycles, this mutation will be amplified with an undiminished efficiency because no further mispaired primings are required.
  • the product having the mutation can be inserted into an appropriate vector by standard techniques for
  • Both the pUC 8 and the pUC 13 plasmlds contain the ampicillin resistance gene to which the primers for the PCR and the amplification method of the present invention (MEA or multiple enzyme amplification) are directed.
  • the innoculated tubes were shaken at 37oC overnight. Growth of the various cultures were scored as follows:
  • each reaction mixture was 50 ⁇ ls.
  • Each reaction was stopped by adding 5 ⁇ ls of 0.5 M EDTA to each tube. 5 ⁇ ls from each reaction was run on a 1% agarose gel along with cesium chloride purified pUC 13 which had been cut with Sea 1 restriction enzyme. The gel was stained with ethidium bromide and scored for the presence of plasmid DNA by eye under ultraviolet light.
  • strain A nor B minipreps was observed to contain plasmids.
  • Both the strain C and D minipreps contained a plasmid with the strain D miniprep plasmid (pUC 13) being present at a much higher concentration than the strain C miniprep plasmid (pUC 8).
  • Each one of the miniprep tubes was diluted by 1000 fold in distilled water and 5 ⁇ ls of that dilution was
  • the sequences of the primers used for both the PCR and MEA reactions were for the promoter primer:
  • These two primers hybridize to two separate regions in the ampicillin resistance gene and are capable of amplifying that gene in either a PCR reaction or MEA reaction, producing a DNA fragment 104 bp in length.
  • the PCR reaction volume was 50 ⁇ ls and had a final concentration of 10 mM Tris pH 8.3, 50 mM KCl, 5 mM MgCl 2 , 1 mM each of dATP, dCTP, dGTP and TTP; 0.2 ⁇ M each primer and 5 units of Taq polymerase (New
  • the PCR reaction was run for 20 cycles consisting of 30 seconds at 95oC, 30 seconds at 37oC, and 3 minutes at 65oC.
  • the MEA reaction was run in a volume of 40 ⁇ ls consisting of 45 mM Tris pH 8.0, 12.5 mM NaCl, 37.5 mM KCl, 1 mM spermidine, 2 mM each of ATP, CTP, GTP, and UTP; 0.5 mM each of dATP, dCTP , dGTP, and TTP; 0.25 ⁇ M each primer, 200 units of M-MLV reverse transcriptase (Bethesda Research Labs), and 60 units of T7 RNA polymerase (New England Biolabs).
  • the reverse transcriptase Bethesda Research Labs
  • T7 RNA polymerase New England Biolabs
  • transcriptase and the T7 RNA polymerase were added after the tubes containing the rest of the reagents were heated to 94oC for 2 minutes and then cooled to 37oC for 30 seconds in order to allow the primers to hybridize to the target DNA.
  • the MEA reaction was allowed to sit at 37oC for 3 hours.
  • approximately 10 molecules of Saul digested cesium chloride pruified pUc13 plasmid was run in the PCR and MEA reactions. This was approximately the same amount of pUC 13 plasmid DNA that was contained in the strain D miniprep as determined by comparing bands on an agarose gel. All reactions were stopped by adding EDTA to a final concentration of 50 mM. 5 ⁇ ls from each reaction was run on a 10% acrylamide gel. The gel was stained with ethidium bromide and scored for the presence of a band corresponding to 104 base pairs by eye.

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Abstract

L'invention concerne un procédé isothermique d'amplification de l'ADN d'intérêt ayant une extrémité 3' définie et contenue dans une séquence ADN cible. Le procédé consiste à combiner, dans des conditions appropriées: la séquence ADN cible traitée, si nécessaire, pour rendre l'ADN d'intérêt disponible à des fins d'hybridation avec des séquences d'acide nucléique complémentaire; Une amorce de promotion; une amorce d'inversion; au moins une polymérase d'ADN dépendante de l'ARN; au moins une polymérase d'ADN dépendante de l'ADN; au moins une polymérase d'ARN dépendante de l'ADN; un agent ayant une activité RNase H; et des triphosphates nucléosides appropriés. De plus, plusieurs modes de réalisation du procédé d'amplification sont décrits. Par exemple, une enzyme de restriction qui reconnait et effectue le clivage spécifiquement soit de l'ADN monobrin soit de l'ADN double brin dans un site sélectionné est également combinée avec l'ADN cible, pour effectuer le clivage de l'ADN cible au niveau d'un site sélectionné et produire l'ADN d'intérêt ayant une extrémité 3' définie.
PCT/US1990/005321 1989-09-20 1990-09-19 Amplification isothermique in vitro de l'acide nucleique WO1991004340A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0587266A1 (fr) * 1992-05-06 1994-03-16 Gen-Probe Incorporated Méthode d'amplification de séquences d'acides nucléiques, composition et trousse d'essai
DE4409436A1 (de) * 1994-03-19 1995-09-21 Boehringer Mannheim Gmbh Verfahren zur Bearbeitung von Nukleinsäuren
WO1996002668A1 (fr) * 1994-07-15 1996-02-01 Azco Nobel N.V. Utilisation d'arn polymerase pour ameliorer un procede d'amplification d'acide nucleique
WO1996017087A1 (fr) * 1994-12-02 1996-06-06 Intelligene Ltd. Detection de sequences d'acide nucleique
WO1997010364A1 (fr) * 1995-09-14 1997-03-20 Digene Diagnostics, Inc. Reaction d'amplification continue
EP0969101A1 (fr) * 1998-07-01 2000-01-05 Tosoh Corporation Procédé de détermination de séquences d'acides nucléiques
JP2001037500A (ja) * 1999-05-24 2001-02-13 Tosoh Corp 核酸定量分析方法
WO2002070735A2 (fr) * 2001-03-07 2002-09-12 Biomerieux B.V. Procede d'amplification et de detection d'adn au moyen d'une amplification fondee sur la transcription
WO2002072881A2 (fr) * 2001-03-07 2002-09-19 Biomerieux B.V. Methode permettant d'amplifier et de detecter un adn hbv par amplification par transcription
US6541205B1 (en) * 1999-05-24 2003-04-01 Tosoh Corporation Method for assaying nucleic acid
WO2004059005A2 (fr) * 2002-10-29 2004-07-15 Guoliang Fu Amplification exponentielle et lineaire combinee
US20050123926A1 (en) * 1994-01-13 2005-06-09 Enzo Diagnostics, Inc., In vitro process for producing multiple nucleic acid copies
US7501254B2 (en) 2006-07-20 2009-03-10 Ghc Technologies, Inc. Methods and compositions for amplification and capture of nucleic acid sequences
CN110358796A (zh) * 2019-07-06 2019-10-22 天津大学 一种基于模板依赖的dna聚合酶的低成本dna合成方法
CN110387399A (zh) * 2018-04-20 2019-10-29 北京全谱医学检验实验室有限公司 一种线性扩增双链dna的方法及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0310229A1 (fr) * 1987-07-31 1989-04-05 The Board Of Trustees Of The Leland Stanford Junior University Amplification sélective de séquences de cible oligonucléotide
WO1990006995A1 (fr) * 1988-12-16 1990-06-28 Siska Diagnostics, Inc. Systeme auto-entretenu de replication sequentielle
EP0329822B1 (fr) * 1988-02-24 1994-06-08 Cangene Corporation Procédé d'amplification d'acides nucléiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0310229A1 (fr) * 1987-07-31 1989-04-05 The Board Of Trustees Of The Leland Stanford Junior University Amplification sélective de séquences de cible oligonucléotide
EP0329822B1 (fr) * 1988-02-24 1994-06-08 Cangene Corporation Procédé d'amplification d'acides nucléiques
WO1990006995A1 (fr) * 1988-12-16 1990-06-28 Siska Diagnostics, Inc. Systeme auto-entretenu de replication sequentielle

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Gene, volume 40, December 1985, Elsevier Science Publishers, W. Szybalski: "Universal restriction endonucleases: designing novel cleavage specificities by combining adapter oligodeoxynucleotide and enzyme moieties", pages 169-173 *
Proc. Natl. Acad. Sci. USA, volume 87, March 1990, J.C. Guatelli et al.: "Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication", pages 1874-1878 *

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US6027897A (en) * 1991-11-14 2000-02-22 Digene Corporation Promoter-primer mediated nucleic acid amplification
US5981179A (en) * 1991-11-14 1999-11-09 Digene Diagnostics, Inc. Continuous amplification reaction
EP0587266A1 (fr) * 1992-05-06 1994-03-16 Gen-Probe Incorporated Méthode d'amplification de séquences d'acides nucléiques, composition et trousse d'essai
US20080026372A9 (en) * 1994-01-13 2008-01-31 Enzo Diagnostics, Inc. In vitro processes for producing multiple copies of primer sequence-free specific nucleic acid
US20050123926A1 (en) * 1994-01-13 2005-06-09 Enzo Diagnostics, Inc., In vitro process for producing multiple nucleic acid copies
DE4409436A1 (de) * 1994-03-19 1995-09-21 Boehringer Mannheim Gmbh Verfahren zur Bearbeitung von Nukleinsäuren
WO1996002668A1 (fr) * 1994-07-15 1996-02-01 Azco Nobel N.V. Utilisation d'arn polymerase pour ameliorer un procede d'amplification d'acide nucleique
US6025134A (en) * 1994-07-15 2000-02-15 Akzo Nobel N.V. Use of RNA polymerase to improve nucleic acid amplification process
WO1996017087A1 (fr) * 1994-12-02 1996-06-06 Intelligene Ltd. Detection de sequences d'acide nucleique
WO1997010364A1 (fr) * 1995-09-14 1997-03-20 Digene Diagnostics, Inc. Reaction d'amplification continue
AU711130B2 (en) * 1995-09-14 1999-10-07 Digene Diagnostics Inc. Continuous amplification reaction
EP0969101A1 (fr) * 1998-07-01 2000-01-05 Tosoh Corporation Procédé de détermination de séquences d'acides nucléiques
US7112407B2 (en) 1998-07-01 2006-09-26 Tosoh Corporation Method of assay of target nucleic acid
US6541205B1 (en) * 1999-05-24 2003-04-01 Tosoh Corporation Method for assaying nucleic acid
JP2001037500A (ja) * 1999-05-24 2001-02-13 Tosoh Corp 核酸定量分析方法
WO2002070735A3 (fr) * 2001-03-07 2003-08-28 Biomerieux Bv Procede d'amplification et de detection d'adn au moyen d'une amplification fondee sur la transcription
WO2002072881A3 (fr) * 2001-03-07 2003-10-30 Biomerieux Bv Methode permettant d'amplifier et de detecter un adn hbv par amplification par transcription
WO2002072881A2 (fr) * 2001-03-07 2002-09-19 Biomerieux B.V. Methode permettant d'amplifier et de detecter un adn hbv par amplification par transcription
WO2002070735A2 (fr) * 2001-03-07 2002-09-12 Biomerieux B.V. Procede d'amplification et de detection d'adn au moyen d'une amplification fondee sur la transcription
KR100948020B1 (ko) * 2001-03-07 2010-03-19 바이오메리욱스 비.브이. 전사 기초한 증폭을 이용하는 ｈｂｖ ｄｎａ의 증폭 및검출 방법
US7723078B2 (en) 2001-03-07 2010-05-25 Biomerieux, B.V. Method for the amplification and detection of HBV DNA using a transcription based amplification
WO2004059005A2 (fr) * 2002-10-29 2004-07-15 Guoliang Fu Amplification exponentielle et lineaire combinee
WO2004059005A3 (fr) * 2002-10-29 2004-08-19 Guoliang Fu Amplification exponentielle et lineaire combinee
AU2003276475B2 (en) * 2002-10-29 2009-11-12 Guoliang Fu Combined exponential and linear amplification
US7501254B2 (en) 2006-07-20 2009-03-10 Ghc Technologies, Inc. Methods and compositions for amplification and capture of nucleic acid sequences
CN110387399A (zh) * 2018-04-20 2019-10-29 北京全谱医学检验实验室有限公司 一种线性扩增双链dna的方法及应用
CN110387399B (zh) * 2018-04-20 2023-04-18 北京全谱医学检验实验室有限公司 一种线性扩增双链dna的方法及应用
CN110358796A (zh) * 2019-07-06 2019-10-22 天津大学 一种基于模板依赖的dna聚合酶的低成本dna合成方法

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