Nicking and Extension Amplification Reaction for the Exponential Amplification of Nucleic Acids

Abstract

The invention is in general directed to the rapid exponential amplification of short DNA or RNA sequences at a constant temperature.

1 - 41 . (canceled) 
     
     
         42 . A kit for amplifying a nucleic acid target sequence, comprising
 a) a DNA polymerase;   b) a first template for nucleic acid amplification, comprising a recognition region of 8-15 nucleotides in length at the 3′ end that is complementary to the 3′ end of a target sequence sense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site;   c) a second template for nucleic acid amplification, comprising a recognition region of 8-15 nucleotides in length at the 3′ end that is complementary to the 3′ end of the complement of the target sequence sense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site; and   d) one or two thermostable nicking enzymes, wherein either one enzyme is capable of nicking at the nicking site of said first and said second templates, or a first enzyme is capable of nicking at the nicking site of said first primer and a second enzyme is capable of nicking at the enzyme site of said second primer.   
     
     
         43 . The kit of  claim 42 , wherein the recognition region of the first template is 8-12 or 8-13 nucleotides in length and the recognition region of the second template is 8-12 or 8-13 nucleotides in length. 
     
     
         44 . The kit of  claim 42 , wherein said polymerase and nicking enzymes are in a first container, and said templates are in a second container. 
     
     
         45 . The kit of  claim 42 , further comprising instructions for following the method of amplification. 
     
     
         46 . The kit of  claim 42 , further comprising a detector component selected from the group consisting of a fluorescent dye, colloidal gold particles, latex particles, a molecular beacon, and polystyrene beads. 
     
     
         47 . The kit of  claim 42 , wherein at least one of said templates comprises a spacer, blocking group, or a modified nucleotide. 
     
     
         48 . The kit of  claim 42 , wherein the stabilizing region has a GC content of at least 40 percent and/or a length of at least 6 nucleotides.