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 method of amplifying a target nucleotide sequence, the method comprising:
 (a) combining a target nucleic acid comprising the target nucleotide sequence with an amplification reagent mixture comprising:
 i. a polymerase, 
 ii. one or more nicking enzymes, 
 iii. a first oligonucleotide comprising a 5′ portion that comprises a nicking site that is non-complementary to the target nucleotide sequence and a 3′ portion that hybridizes to the target nucleotide sequence, and 
 iv. a second oligonucleotide comprising a 5′ portion that comprises a nicking site that is non-complementary to the target nucleotide sequence and a 3′ portion that hybridizes to the target nucleotide sequence, and 
   (b) subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions to amplify the target nucleotide sequence;   wherein:   the target nucleotide sequence is between 20 and 40 nucleotides in length; and   (ii) the target nucleotide sequence is amplified 1E+6-fold or more in about in about ten minutes.   
     
     
         43 . The method of  claim 42 , wherein no oligonucleotides other than the first oligonucleotide and the second oligonucleotide are necessary to amplify the target nucleotide sequence. 
     
     
         44 . The method of  claim 42 , wherein the amplification reagent mixture contains no oligonucleotides other than the first oligonucleotide and the second oligonucleotide. 
     
     
         45 . The method of  claim 42 , wherein the target nucleic acid is present in a sample obtained from an animal, plant or food. 
     
     
         46 . The method of  claim 42 , wherein the target nucleic acid is not subjected to a thermal denaturation step prior to the step of combining. 
     
     
         47 . The method of  claim 42 , wherein the step of combining occurs in a single step. 
     
     
         48 . The method of  claim 45 , wherein the target nucleic acid is present in a sample obtained from an animal and the obtained sample is diluted prior to the step of combining. 
     
     
         49 . The method of  claim 42 , wherein the amplification reagent mixture is free of bumper primers. 
     
     
         50 . The method of  claim 42 , wherein the target nucleotide sequence is amplified without the assistance of bumper primers. 
     
     
         51 . The method of  claim 42 , further comprising (c) detecting the amplified target nucleotide sequence within about 10 minutes or less of subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions. 
     
     
         52 . The method of  claim 51 , wherein (c) detecting the amplified target nucleotide sequence occurs in real time. 
     
     
         53 . The method of  claim 45 , wherein the sample is obtained from an animal and the animal is a human, the target nucleic acid is a target nucleic acid of a human pathogen, and the sample is obtained from the mucus, sputum, or saliva of the human. 
     
     
         54 . The method of  claim 53 , wherein the human pathogen is a single-stranded DNA virus, double-stranded DNA virus, or a single-stranded RNA virus. 
     
     
         55 . The method of  claim 53 , wherein the human pathogen is a bacterium. 
     
     
         56 . The method of  claim 42 , wherein the first oligonucleotide further comprises a nicking enzyme binding site and a stabilizing region. 
     
     
         57 . The method of  claim 42 , wherein the second oligonucleotide further comprises a nicking enzyme binding site and a stabilizing region. 
     
     
         58 . The method of  claim 56 , wherein the one or more nicking enzymes nicks downstream of the nicking enzyme binding site. 
     
     
         59 . The method of  claim 57 , wherein the one or more nicking enzymes nicks downstream of the nicking enzyme binding site. 
     
     
         60 . The method of  claim 42 , wherein each of the first oligonucleotide and the second oligonucleotide comprises a nicking enzyme binding site recognized by the same nicking enzyme and the one or more nicking enzymes are the same. 
     
     
         61 . The method of  claim 42 , wherein the 3′ portion of the first oligonucleotide that hybridizes to the target nucleotide sequence is 8-15 nucleotides long. 
     
     
         62 . The method of  claim 42 , wherein the 3′ portion of the second oligonucleotide that hybridizes to the target nucleotide sequence is 8-15 nucleotides long. 
     
     
         63 . The method of  claim 42 , wherein the target nucleic acid is double-stranded DNA. 
     
     
         64 . The method of  claim 42 , wherein the target nucleic acid is single-stranded DNA. 
     
     
         65 . The method of  claim 42 , wherein the target nucleic acid is RNA. 
     
     
         66 . The method of  claim 42 , wherein the target nucleic acid is selected from the group consisting of genomic DNA, plasmid DNA, viral DNA, mitochondrial DNA, cDNA, synthetic double-stranded DNA and synthetic single-stranded DNA. 
     
     
         67 . The method of  claim 42 , wherein the target nucleic acid is selected from the group consisting of messenger RNA, viral RNA, ribosomal RNA, transfer RNA, micro RNA, micro RNA precursor, and synthetic RNA. 
     
     
         68 . The method of  claim 42 , wherein the temperature of the essentially isothermal conditions is between 54° C. and 60° C. 
     
     
         69 . The method of  claim 42 , wherein the temperature of the essentially isothermal conditions is between 56° C. and 58° C. 
     
     
         70 . The method of  claim 42 , wherein at least two different target nucleotide sequences are amplified. 
     
     
         71 . The method of  claim 42 , wherein the target nucleotide sequence is amplified 1E+6-fold or more in about five minutes. 
     
     
         72 . The method of  claim 42 , wherein the target nucleotide sequence is amplified 1E+6-fold or more in about 2.5 minutes. 
     
     
         73 . The method of  claim 42 , wherein the target nucleotide sequence is amplified 1E+7-fold or more in about five minutes. 
     
     
         74 . The method of  claim 42 , wherein the target nucleotide sequence is amplified 1E+8-fold or more in about five minutes. 
     
     
         75 . The method of  claim 42 , wherein the target nucleotide sequence is amplified 1E+9-fold or more in about five minutes. 
     
     
         76 . A system configured to amplify a target nucleotide sequence under essentially isothermal conditions and comprising an amplification reagent mixture, the amplification reagent mixture comprising:
 i. a polymerase,   ii. one or more nicking enzymes,   iii. a first oligonucleotide comprising a 5′ portion that comprises a nicking site that is non-complementary to the target nucleotide sequence and a 3′ portion that hybridizes to the target nucleotide sequence, and   iv. a second oligonucleotide comprising a 5′ portion that comprises a nicking site that is non-complementary to the target nucleotide sequence and a 3′ portion that hybridizes to the target nucleotide sequence;
 and wherein the target nucleotide sequence is between 20 and 40 nucleotides in length. 
   
     
     
         77 . The system of  claim 76 , further configured to amplify the target nucleotide sequence 1E+6-fold or more in about in about ten minutes. 
     
     
         78 . The system of  claim 76 , wherein the amplification reagent mixture contains no oligonucleotides other than the first oligonucleotide and the second oligonucleotide. 
     
     
         79 . The system of  claim 76 , further configured to detect the amplified target nucleotide sequence. 
     
     
         80 . The system of  claim 79 , wherein detecting the amplified target nucleotide sequence occurs within about 10 minutes or less of combining a target nucleotide sequence with the amplification reagent mixture under essentially isothermal conditions. 
     
     
         81 . The system of  claim 76 , wherein detecting amplified target nucleotide sequence occurs in real time. 
     
     
         82 . The system of  claim 76 , further configured to amplify a target nucleic acid present in a sample from an animal, plant or food. 
     
     
         83 . The system of  claim 76 , further configured to amplify a target nucleic acid of a human pathogen present in a sample of mucus, sputum, or saliva obtained from a human.