Enhanced nucleic acid identification and detection
The present invention relates to assays, including amplification assays, conducted in the presence of modulators. These assays can be used to detect the presence of particular nucleic acid sequences. In particular, these assays can allow for genotyping or other genetic analysis.
1. A method, comprising:
providing a volume suspected of containing target nucleic acids;
dispersing said volume among a plurality of areas, such that said plurality of areas comprises a distribution of nucleic acids said distribution capable of generating digital nucleic acid amplification signals; and
conducting a Loop-mediated isothermal nucleic acid amplification (LAMP) reaction in said plurality of areas in the presence of a restriction enzyme, wherein said restriction enzyme modulates said isothermal nucleic acid amplification in the presence of said target nucleic acids and wherein said restriction enzyme acts on said target nucleic acids within a region amplified by said isothermal nucleic acid amplification reaction,
wherein the LAMP reaction comprises an F3 primer and a B3 primer and the restriction enzyme recognizes a restriction site that occurs in a region of said target nucleic acid that is between the B3 and F3 primer.
2. The method of claim 1 , wherein within said distribution, said plurality of areas each comprise at most one of said target nucleic acids.
3. The method of claim 1 , wherein said modulation comprises producing a difference in amplification efficiency.
4. The method of claim 3 , wherein said difference in amplification efficiency produces a positive amplification signal in a subset of said plurality of areas.
5. The method of claim 4 , wherein said positive amplification signal in said subset of said plurality of areas is diagnostic of the presence of one or more distinct target nucleic acids within said volume.
6. The method of claim 1 , wherein the target nucleic acids comprises an HCV nucleic acid.
7. The method of claim 6 , wherein said method generates a signal from which an HCV genotype can be determined.
8. The method of claim 1 , further comprising comparing results of said isothermal nucleic acid amplification reaction to results of a control isothermal nucleic acid amplification reaction carried out in the absence of the modulator.
9. A method, comprising:
providing a volume suspected of containing target nucleic acids;
dispersing said volume among a plurality of areas, such that said plurality of areas comprises a distribution of nucleic acids said distribution capable of generating digital nucleic acid amplification signals; and
conducting an isothermal NASBA nucleic acid amplification reaction in said plurality of areas in the presence of a restriction enzyme, wherein said restriction enzyme modulates said isothermal nucleic acid amplification in the presence of said target nucleic acids and wherein said restriction enzyme acts on said target nucleic acids within a region amplified by said isothermal nucleic acid amplification reaction,
wherein the NASBA reaction comprises a forward primer and a reverse primer and the restriction enzyme recognizes a restriction site that occurs in a region of the target nucleic acid that is between the forward primer and the reverse primer, including the priming region.
10. The method of claim 9 , wherein within said distribution, said plurality of areas each comprise at most one of said target nucleic acids.
11. The method of claim 9 , wherein said modulation comprises producing a difference in amplification efficiency.
12. The method of claim 11 , wherein said difference in amplification efficiency produces a positive amplification signal in a subset of said plurality of areas.
13. The method of claim 9 , wherein the target nucleic acids comprise an HCV nucleic acid.
14. The method of claim 13 , wherein said method generates a signal from which an HCV genotype can be determined.
15. The method of claim 9 , further comprising comparing results of said isothermal nucleic acid amplification reaction to results of a control isothermal nucleic acid amplification reaction carried out in the absence of the modulator.