Endonuclease-enhanced helicase-dependent amplification
View Patent ↗The invention provides methods and compositions for enhancing the speed and sensitivity of helicase-dependent amplification through the use of an endonuclease.
1. A method for assaying a sample, the method comprising combining a composition comprising a restriction endonuclease, a helicase, a DNA polymerase, and at least a first primer and a second primer,
with a sample containing or potentially containing a nucleic acid that hybridizes to the first and second primer, and
amplifying the target sequence to generate an amplification product
wherein the restriction endonuclease is one which cannot recognize or cleave the amplification product.
2. The method of claim 1 , wherein the sample is a biological sample from a subject.
3. The method of claim 1 , wherein the sample comprises human tissue, cells or body fluid.
4. The method of claim 1 , wherein the sample is an environmental, animal or industrial sample.
5. The method of claim 1 , wherein the sample comprises no more than 1 ng of DNA.
6. The method of claim 5 , wherein the sample comprises no more than 0.01 ng of DNA.
7. The method of claim 1 , wherein the sample comprises no more than about 300 copies of the nucleic acid that hybridizes to the first and second primer.
8. The method of claim 7 , wherein the sample comprises no more than about 3 copies of the nucleic acid that hybridizes to the first and second primer.
9. A method for amplifying a nucleic acid, the method comprising the steps of:
(a) treating the nucleic acid with a restriction endonuclease,
(b) incubating the nucleic acid with at least a first primer and a second primer and amplifying the nucleic acid in a helicase-dependent amplification reaction to generate the amplified product, wherein the amplified product is not cleaved by the restriction endonuclease.
10. The method of claim 9 , wherein step (a) is performed prior to step (b).
11. The method of claim 9 , wherein steps (a) and (b) are performed simultaneously.
12. The method of claim 9 , wherein the restriction endonuclease is a Type II restriction endonuclease.
13. The method of claim 9 , wherein the restriction endonuclease is a Type IIs restriction endonuclease.
14. The method of claim 9 , wherein the restriction endonuclease is AvaII, BanII, AseI, SspI, SpeI, MboI, MboII, SwaI, BsrI, BtsCI, CviQI, CviAII, Tsp45I, Fnu4HI, MsII, MspAII, SacII, AgeI, DraI, or XmnI.
15. The method of claim 9 , wherein the helicase-dependent amplification reaction comprises a helicase, a DNA polymerase, a buffer, deoxynucleotides, magnesium, and at least the first and second primer.
16. The method of claim 15 , wherein the helicase is a thermostable helicase.
17. The method of claim 1 , wherein the restriction endonuclease cleaves the nucleic acid within 5 kb of a sequence that hybridizes to the first and/or second primer.
18. The method of claim 17 , wherein the restriction endonuclease cleaves the nucleic acid within 500 bb of a sequence that hybridizes to the first and/or second primer.