Endonuclease-enhanced helicase-dependent amplification

Abstract

The invention provides methods and compositions for enhancing the speed and sensitivity of helicase-dependent amplification through the use of an endonuclease.

We claim: 
     
         1 . A kit for amplifying a target nucleic acid sequence in a sample, comprising
 a type II restriction endonuclease, a helicase, a DNA polymerase, and at least a first primer and a second primer,   wherein the sample may or may not contain a DNA molecule 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; and   wherein the type II restriction endonuclease is one which cannot recognize or cleave the amplification product.   
     
     
         2 . The kit according to  claim 1 , wherein the type II restriction endonuclease is one which cleaves the target DNA within 5000 nucleotides of the target nucleic acid. 
     
     
         3 . The kit according to  claim 1 , wherein the type II restriction endonuclease is one which can recognize and cleave the DNA within 500 nucleotides of the target nucleic acid. 
     
     
         4 . The kit according to  claim 1 , wherein the helicase is a thermostable helicase. 
     
     
         5 . The kit according to  claim 1 , further comprising a buffer. 
     
     
         6 . The kit according to  claim 1 , further comprising deoxynucleotides. 
     
     
         7 . The kit according to  claim 1 , further comprising magnesium. 
     
     
         8 . A method for detecting a DNA molecule comprising a target nucleic acid in a sample, the method comprising
 (a) treating the DNA molecule with a restriction endonuclease;   (b) incubating the DNA molecule with at least a first primer and a second primer and amplifying the nucleic acid in a helicase-dependent amplification reaction to generate an amplified product, wherein the amplified product is not cleaved by the restriction endonuclease.   
     
     
         9 . The method of  claim 8 , wherein step (a) is performed prior to step (b). 
     
     
         10 . The method of  claim 8 , wherein step (a) and step (b) are performed simultaneously. 
     
     
         11 . The method of  claim 8 , wherein the restriction endonuclease is a type II restriction endonuclease. 
     
     
         12 . The method of  claim 8 , wherein the restriction endonuclease is a type IIs restriction endonuclease. 
     
     
         13 . The method of  claim 8 , wherein the restriction endonuclease cleaves the nucleic acid within 5000 nucleotides of a sequence that hybridizes to the first and/or second primer. 
     
     
         14 . The method of  claim 8 , wherein the restriction endonuclease cleaves the nucleic acid within 500 nucleotides of a sequence that hybridizes to the first and/or second primer. 
     
     
         15 . The method of  claim 8 , wherein the helicase-dependent amplification reaction comprises a helicase, a DNA polymerase, a buffer, and the at least first and second primers. 
     
     
         16 . The method of  claim 15 , wherein the helicase-dependent amplification reaction further comprises magnesium. 
     
     
         17 . The method of  claim 15 , wherein the helicase-dependent amplification reaction further comprises deoxynucleotides.