Method for enhancing amplification of a target nucleic acid sequence
View Patent ↗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.
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.
2. The method of claim 1 , wherein amplification oligonucleotides are not provided to said mixture prior to step b).
3. The method of claim 2 , wherein said inhibitor does not hybridize to said target nucleic acid under said amplification conditions to form a stable inhibitor:target complex.
4. The method of claim 3 , wherein said amplification oligonucleotides and said reversible inhibitor compete for binding said RNA polymerase.
5. The method of claim 1 , wherein step c) is carried out under isothermal conditions.
6. The method of claim 1 , wherein said inhibitor is a decoy probe comprising a nucleotide base recognition sequence region which binds to said RNA polymerase.
7. The method of claims 6 , wherein the nucleotide base sequence region of said decoy probe has at least 35% sequence similarity to a promoter sequence recognized by said RNA polymerase.
8. The method of claim 6 , wherein said decoy probe comprises:
a first nucleotide base recognition sequence region, wherein said first region binds to an RNA polymerase; and
an optionally present second nucleotide base recognition sequence region,
provided that if said first region is nucleic acid and said second region is present, then said second region is either directly joined to the 5′ end of said first region or is joined to the 3′ end or 5′ end of said first region by a non-nucleotide linker, wherein said optionally present second region is present if said first region can be used to produce a functional double-stranded promoter sequence using a complementary oligonucleotide,
further provided that if said first region is nucleic acid which can be used to produce said functional double-stranded promoter sequence using said complementary oligonucleotide, then said decoy probe does not have a nucteic acid sequence greater than about 10 nucleotides in length joined directly to the 3′ end of said first region and said decoy probe does not have a terminal 3′ OH group available to accept a nucleoside triphosphate in a polymerization reaction.
9. The method of claim 8 , wherein said first region is nucleic acid, said second region is present and joined directly to the 5′ end of said first region, and said probe does not have a nucleic acid sequence greater than 10 nucleotides in length joined directly to its 3′ end.
10. The method of claim 8 , wherein said first region is nucleic acid, said second region is present and joined to the 3′ end or 5′ end of said first region by a non-nucleotide linker, and said probe does not have a nucleic acid sequence greater than 10 nucleotides in length joined directly to its 3′ end.
11. The method of claim 8 , wherein said first region does not have a nucleic acid sequence greater than 5 nucleotides in length joined directly to its 3′ end.
12. The method of claim 8 , wherein said second region is present and includes a region of self-complementarity which can form a hairpin structure.
13. The method of claim 8 , wherein said probe does not include a nucleotide base sequence which is substantially complementary to a nucleotide base sequence present in said target nucleic acid or a complement thereof.
14. The method of claim 8 , wherein said probe consists of 15 to 100 optionalty modified nucleosides and one or more blocking groups located at each 3′ terminus of said probe, wherein each of said optionally modified nucleosides independently includes:
a purine or pyrirnidine moiety independently selected from the group consisting of inosine, uracil, adenine, guanine, thymine and cytosine; and
a sugar moiety independently selected from the group consisting of deoxyribose, 2′-methoxy ribose, and ribose,
wherein each of said optionally modified nucleosides is joined together by an internucleoside linkage independently selected from the group consisting of phosphodiester, phosphorothioate, and methylphosphonate.
15. The method of claim 14 , wherein at least 80% of said optionally modified nucleosides have a purine or pyrimidine moiety independently selected from the group consisting of adenine, guanine, thymine and cytosine, and a deoxyribose sugar moiety, and wherein at least 80% of said internucleoside linkages joining said optionally modified nucleosides are phosphodiester.
16. The method of claim 15 , wherein said probe consists of 15 to 100 independently selected deoxyribonucleotides and one or more blocking groups located at each 3′ terminus of said probe.
17. The method of claim 16 , wherein said one or more blocking groups are selected from the group consisting of phosphorothioate, alkane-diol residue, cordycepin, and an alkyl group.
18. The method of claim 16 , wherein said probe consists of 35 to 70 independently selected nucleotides and said one or more blocking groups, and wherein said second region is present and comprises at least 10 nucleotides.
19. The method of claim 8 , wherein said RNA polymerase is a T3, T7 or SP6 RNA polymerase.
20. The method of claim 19 , wherein said first region has at least 35% sequence similarity to an RNA polymerase promoter sequence.
21. The method of claim 20 , wherein said first region has a nucleotide base sequence similarity of at least 75% to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
22. The method claim 21 , wherein said first region has a nucleotide base sequence similarity of 75% to 95% to the sequence of SEQ ID NO: 3.
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:
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.
24. The method of claim 23 , wherein said DNA polymerase is a reverse transcriptase and said means for reversibly inhibiting said RNA polymerase is not a substrate in a primer extension reaction.
25. The method of claim 24 , wherein said RNA polymerase, said reverse transcriptase, and said means for reversibly inhibiting said RNA polymerase are first combined together in the absence of said promoter-template complementary oligonucleotide.
26. The method of claim 23 , wherein said promoter-template complementary oligonucleotide and said means for reversibly inhibiting said RNA polymerase compete for binding said RNA polymerase.
27. The method of claim 23 , wherein said amplifying step is carried out under isothermal conditions.
28. A method useful for amplifying a target nucleic acid sequence comprising the steps of:
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.
29. The method of claim 28 , wherein amplification oligonucleotides are not provided to said mixture prior to step b).
30. The method of claim 28 , wherein said inhibitor does not form a stable inhibitor:target complex under said isothermal amplification conditions.