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 composition for amplifying a target nucleic acid sequence in a sample, comprising
a type II restriction endonuclease, a helicase, a DNA polymerase, a DNA molecule comprising the target nucleic acid sequence, and at least a first primer and a second primer, wherein the DNA molecule is bound by the type II restriction endonuclease, the helicase, the DNA polymerase and the at least first and second primers;
wherein the sample may or may not contain DNA which comprises the target nucleic acid;
wherein the target nucleic acid is one which can hybridizes to at least the first and second primer and is amplified in the presence of the composition to generate an amplification product wherein the 5′ end of the amplification product is defined by the 5′ end of the first primer and the 3′ end of the amplification product is defined by the 5′ end of the second primer;
wherein the type II restriction endonuclease is one which does not recognize or cleave the amplification product; and
wherein the type II restriction endonuclease is one which cleaves the target DNA within 5000 bases of the target nucleic acid.
2. The composition of claim 1 , wherein the restriction endonuclease is a Type IIs restriction endonuclease.
3. The composition of claim 1 , 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.
4. The composition of claim 1 , wherein the helicase is a thermostable helicase.
5. The composition of claim 1 , wherein the composition further comprises deoxynucleotides.
6. The composition of claim 1 , wherein the composition further comprises a buffer.
7. The composition of claim 1 , wherein the composition further comprises magnesium.
8. A kit for amplifying a target nucleic acid sequence in a sample, comprising
a type II restriction endonuclease, a helicase, a DNA polymerase, a DNA molecule comprising the target nucleic acid sequence, and at least a first primer and a second primer,
wherein the DNA molecule is bound by the at least type II restriction endonuclease, the helicase, the DNA polymerase and the first and second primers
wherein the sample may or may not contain DNA which comprises the target nucleic acid;
wherein the target nucleic acid is one which can hybridize to at least the first and second primer and is amplified in the presence of the composition to generate an amplification product wherein the 5′ end of the amplification product is defined by the 5′ end of the first primer and the 3′ end of the amplification product is defined by the 5′ end of the second primer;
wherein the type II restriction endonuclease is one which cannot recognize or cleave the amplification product; and
wherein the type II restriction endonuclease is one which cleaves the target DNA within 5000 bases of the target nucleic acid.
9. The kit of claim 8 , wherein the restriction endonuclease is a Type IIs restriction endonuclease.
10. The kit of claim 8 , 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.
11. The kit of claim 8 , wherein the helicase is a thermostable helicase.
12. The kit of claim 8 , wherein the kit further comprises deoxynucleotides.
13. The kit of claim 8 , wherein the kit further comprises a buffer.
14. The kit of claim 8 , wherein the kit further comprises magnesium.
15. The composition of claim 1 , wherein the type II restriction endonuclease is one which can recognize and cleave the DNA within 500 bases of the target nucleic acid.
16. The composition of claim 8 , wherein the type II restriction endonuclease is one which can recognize and cleave the DNA within 500 bases of the target nucleic acid.