Method for nucleic acid amplification

Abstract

A kit for amplifying a nucleic acid, using RNA as a template, which can realize elimination of the risk of non-specific amplification caused by DNA mixed from reagents and/or working environment, an increase in the detection sensitivity of trace RNA, and a reduction in amplification bias. The kit includes a degrading enzyme specific to DNA in an RNA-DNA hybrid that is a double strand-specific DNA degrading enzyme or a non-specific DNA degrading enzyme, and an RNase H minus reverse transcriptase. If the degrading enzyme specific to DNA in an RNA-DNA hybrid is a non-specific DNA degrading enzyme, then the kit further comprises a single-stranded DNA-binding protein.

1 . A kit, comprising:
 a degrading enzyme specific to DNA in an RNA-DNA hybrid, and   an RNase H minus reverse transcriptase, wherein:   the degrading enzyme specific to DNA in an RNA-DNA hybrid is a double strand-specific DNA degrading enzyme or a non-specific DNA degrading enzyme, and   if the degrading enzyme specific to DNA in an RNA-DNA hybrid is a non-specific DNA degrading enzyme, then the kit further comprises a single-stranded DNA-binding protein.   
     
     
         2 . The kit according to  claim 1 , wherein the double strand-specific DNA degrading enzyme is a Crustacea-derived double strand-specific DNA degrading enzyme or a variant thereof. 
     
     
         3 . The kit according to  claim 1  wherein the double strand-specific DNA degrading enzyme is selected from the group consisting of  Solenocera melantho  (coastal mud shrimp) DNase,  Penaeus japonicus  (prawn) DNase,  Paralithodes camtschaticus  (king crab) DSN,  Pandalus borealis  (Northern shrimp) dsDNase,  Chionoecetes opilio  (snow crab) DSN, and other DSN homologs. 
     
     
         4 . The kit according to  claim 1 , wherein the single-stranded DNA-binding protein is T4 gene 32 protein. 
     
     
         5 . The kit according to  claim 1 , further comprising a substrate. 
     
     
         6 . The kit according to  claim 1 , further comprising a primer. 
     
     
         7 . The kit according to  claim 5 , further comprising a primer.