Method for enhancing amplification of a target nucleic acid sequence
First Claim
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1. A method useful for amplifying a target nucleic acid sequence comprising the steps of:
- a) producing a mixture comprising an RNA polymerase and a reversible inhibitor of said RNA polymerase, wherein said reversible inhibitor does not hybridize to a target nucleic acid comprising said target nucleic acid sequence under transcription-associated amplification conditions, and wherein said mixture does not contain said target nucleic acid;
b) combining said mixture and a sample suspected of containing said target nucleic acid; and
c) exposing the combination of step b) to said amplification conditions, such that said target nucleic acid sequence, if present in said sample, is amplified.
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Abstract
The present invention features inhibitors of target-independent amplification and the use of such inhibitors for enhancing an amplification protocol. The inhibitors are believed to enhance an amplification protocol by inhibiting the ability of one or more nucleic acid polymerases to use nucleic acid in a polymerase reaction in the absence of target nucleic acid.
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Citations
30 Claims
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1. A method useful for amplifying a target nucleic acid sequence comprising the steps of:
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a) producing a mixture comprising an RNA polymerase and a reversible inhibitor of said RNA polymerase, wherein said reversible inhibitor does not hybridize to a target nucleic acid comprising said target nucleic acid sequence under transcription-associated amplification conditions, and wherein said mixture does not contain said target nucleic acid; b) combining said mixture and a sample suspected of containing said target nucleic acid; and c) exposing the combination of step b) to said amplification conditions, such that said target nucleic acid sequence, if present in said sample, is amplified. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A transcription-associated amplification procedure comprising the step of amplifying a target nucleic acid sequence to produce multiple copies of RNA transcripts by combining together under transcription-associated amplification cionditions a mixture comprising:
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a) a target nucleic acid comprising said target nucleic acid sequence, a promoter-template complementary oligonucleotide, a DNA polymerase, an RNA polymerase, ribonucleoside triphosphates, deoxyribonucleoside triphosphates; and b) means for reversibly inhibiting said RNA polymerase, wherein said means for reversibly inhibiting said RNA polymerase does not hybridize to said target nucleic acid under said amplification conditions to form a stable inhibitor;
target complex. - View Dependent Claims (24, 25, 26, 27)
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28. A method useful for amplifying a target nucleic acid sequence comprising the steps of:
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a) producing a mixture comprising an amplification enzyme and a reversible inhibitor of said enzyme, wherein said mixture does not contain a target nucleic acid comprising said target nucleic acid sequence; b) combining said mixture and a sample suspected of containing said target nucleic acid; and c) exposing the combination of step b) to conditions, sufficient to amplify said target nucleic acid sequence by strand displacement amplification, wherein said reversible inhibitor does not hybridize to said target nucleic acid under said amplification conditions. - View Dependent Claims (29, 30)
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Specification