Strand displacement amplification
First Claim
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1. A method for amplifying a target nucleic acid sequence comprising:
- a) providing a single stranded nucleic acid fragment containing the target nucleic acid sequence, the fragment having a 5'"'"' end and a 3'"'"' end;
b) binding an oligonucleotide primer to the 3'"'"' end of the fragment such that the primer forms a 5'"'"' single stranded overhang, the primer comprising a 3'"'"' end complementary to the 3'"'"' end of the fragment and a 5'"'"' end comprising a recognition sequence for a restriction endonuclease which does not cut the target nucleic acid sequence;
c) extending the primer on the fragment in the presence ofi) a DNA polymerase lacking 5'"'"'-3'"'"' exonuclease activity,ii) deoxynucleoside triphosphates,iii) at least one substituted deoxynucleoside triphosphate, andiv) a restriction endonuclease which nicks the recognition sequence when the recognition sequence is double stranded and hemimodified by incorporation of the substituted deoxynucleoside triphosphate, thereby producing a double stranded first reaction product comprising the primer, a first newly synthesized strand and a hemimodified restriction endonuclease recognition sequence;
d) nicking the double stranded hemimodified restriction endonuclease recognition sequence with the restriction endonuclease;
e) extending from the nick using the polymerase, thereby displacing the first newly synthesized strand from the first reaction product and generating a second newly synthesized strand, and;
f) repeating the nicking, extending and displacing steps such that the target sequence is amplified.
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Abstract
This invention relates a nucleic acid target amplification and detection method which operates at a single temperature and makes use of a polymerase in conjunction with an endonuclease that will nick the polymerized strand such that the polymerase will displace the strand without digestion while generating a newly polymerized strand.
174 Citations
23 Claims
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1. A method for amplifying a target nucleic acid sequence comprising:
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a) providing a single stranded nucleic acid fragment containing the target nucleic acid sequence, the fragment having a 5'"'"' end and a 3'"'"' end; b) binding an oligonucleotide primer to the 3'"'"' end of the fragment such that the primer forms a 5'"'"' single stranded overhang, the primer comprising a 3'"'"' end complementary to the 3'"'"' end of the fragment and a 5'"'"' end comprising a recognition sequence for a restriction endonuclease which does not cut the target nucleic acid sequence; c) extending the primer on the fragment in the presence of i) a DNA polymerase lacking 5'"'"'-3'"'"' exonuclease activity, ii) deoxynucleoside triphosphates, iii) at least one substituted deoxynucleoside triphosphate, and iv) a restriction endonuclease which nicks the recognition sequence when the recognition sequence is double stranded and hemimodified by incorporation of the substituted deoxynucleoside triphosphate, thereby producing a double stranded first reaction product comprising the primer, a first newly synthesized strand and a hemimodified restriction endonuclease recognition sequence; d) nicking the double stranded hemimodified restriction endonuclease recognition sequence with the restriction endonuclease; e) extending from the nick using the polymerase, thereby displacing the first newly synthesized strand from the first reaction product and generating a second newly synthesized strand, and; f) repeating the nicking, extending and displacing steps such that the target sequence is amplified. - View Dependent Claims (2, 3)
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4. A method for amplifying a target nucleic acid sequence comprising:
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a) providing a double stranded nucleic acid fragment containing the target nucleic acid sequence; b) denaturing the double stranded fragment to produce a first single stranded fragment having a 5'"'"' end and a 3'"'"' end and a second single stranded fragment having a 5'"'"' end and a 3'"'"' end; c) binding a first oligonucleotide primer to the 3'"'"' end of the first single stranded fragment and a second oligonucleotide primer to the 3'"'"' end of the second single stranded fragment such that the first and second primers form 5'"'"' single stranded overhangs on the first and second single stranded fragments, respectively, the first and second primers each comprising a 3'"'"' end complementary to the 3'"'"' end of the first or second single stranded fragment and a 5'"'"' end comprising a recognition sequence for a restriction endonuclease which does not cut the target nucleic acid sequence; d) extending the first and second primers on the first and second single stranded fragments, respectively, in the presence of i) a DNA polymerase lacking 5'"'"'-3'"'"' exonuclease activity, ii) deoxynucleoside triphosphates, iii) at least one substituted deoxynucleoside triphosphate, and iv) a restriction endonuclease which nicks the recognition sequence when the recognition sequence is double stranded and hemimodified by incorporation of the substituted deoxynucleoside triphosphate, thereby producing a double stranded first reaction product comprising the first primer, a first newly synthesized strand and a first hemimodified restriction endonuclease recognition sequence and a double stranded second reaction product comprising the second primer, a second newly synthesized strand and a second hemimodified restriction endonuclease recognition sequence; e) nicking the first and second hemimodified restriction endonuclease recognition sites with the restriction endonuclease; f) extending from the nicks using the polymerase, thereby displacing the first newly synthesized strand from the first reaction product and displacing the second newly synthesized strand from the second reaction product and generating a third and a fourth newly synthesized strand, respectively; g) repeating the nicking, extending and displacing steps such that the target sequence is amplified. - View Dependent Claims (5, 6, 7, 8, 9, 10)
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11. A method for amplifying a target nucleic acid sequence comprising:
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a) providing a single stranded nucleic acid fragment containing the target nucleic acid sequence, the fragment having a 5'"'"' end and a 3'"'"' end; b) binding an oligonucleotide primer to the 3'"'"' end of the fragment such that the primer forms a 5'"'"' single stranded overhang, the primer comprising a 3'"'"' end complementary to the 3'"'"' end of the fragment and a 5'"'"' end comprising a recognition sequence for a restriction endonuclease which does not cut the target nucleic acid sequence; c) extending the primer on the fragment in the presence of i) a DNA polymerase lacking 5'"'"'-3'"'"' exonuclease activity, ii) deoxynucleoside triphosphates, iii) at least one methyl-substituted deoxynucleoside triphosphate, and iv) a restriction endonuclease which nicks the recognition sequence when the recognition sequence is double-stranded and hemimodified by incorporation of the methyl-substituted deoxynucleoside triphosphate, thereby producing a double stranded first reaction product comprising the primer, a first newly synthesized strand and a hemimodified restriction endonuclease recognition sequence; d) nicking the double stranded hemimodified restriction endonuclease recognition sequence with the restriction endonuclease; e) extending from the nick using the polymerase, thereby displacing the first newly synthesized strand from the first reaction product and generating a second newly synthesized strand, and; f) repeating the nicking, extending and displacing steps such that the target sequence is amplified. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method for amplifying a target nucleic add sequence comprising:
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a) providing a single stranded nucleic acid fragment containing the target nucleic acid sequence, the fragment having a 5'"'"' end and a 3'"'"' end; b) binding an oligonucleotide primer to the 3'"'"' end of the fragment such that the primer forms a 5'"'"' single stranded overhang, the primer comprising a 3'"'"' end complementary to the 3'"'"' end of the fragment and a 5'"'"' end comprising a recognition sequence for a restriction endonuclease which does not cut the target nucleic acid sequence; c) extending the primer on the fragment in the presence of i) a DNA polymerase lacking 5'"'"'-3'"'"' exonuclease activity, ii) deoxynucleoside triphosphates, iii) at least one substituted deoxynucleoside triphosphate, and iv) a restriction endonuclease which nicks the recognition sequence when the recognition sequence is double stranded and hemimodified by incorporation of the substituted deoxynucleoside triphosphate, thereby producing a double stranded first reaction product comprising a hemimodified restriction endonuclease recognition sequence; d) nicking the double stranded hemimodified restriction endonuclease recognition sequence with the restriction endonuclease; e) extending from the nick using the polymerase, thereby displacing a copy of the target sequence, and; f) repeating the nicking, extending and displacing steps such that the target sequence is amplified. - View Dependent Claims (22, 23)
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Specification