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.

What is claimed is: 
     
         1 - 66 . (canceled) 
     
     
         67 . A method for amplifying a double-stranded nucleic acid target sequence, comprising:
 a) contacting a target DNA molecule comprising a double-stranded target sequence having a sense strand and an antisense strand with: (1) a first oligonucleotide and a second oligonucleotide, wherein i) said first oligonucleotide comprises a nucleic acid sequence comprising a recognition region at its 3′ end that is complementary to the target sequence antisense strand; a first nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site, wherein the portion of the nucleic acid sequence that is complementary to the target sequence strand is 8-15 nucleotides in length; wherein when the recognition region of the forward oligonucleotide is hybridized to the target sequence, the stabilizing region of the forward oligonucleotide is not hybridized to the target nucleic acid molecule; ii) said second oligonucleotide comprises a nucleotide sequence comprising a recognition region at its 3′ end that is complementary to the target sequence sense strand; a nicking enzyme binding site and a second nicking site upstream of said recognition region, and a stabilizing region upstream of said nicking site, wherein the portion of the nucleic acid sequence that is complementary to the target sequence strand is 8-15 nucleotides in length; wherein when the recognition region of the reverse oligonucleotide is hybridized to the target sequence, the stabilizing region of the reverse oligonucleotide is not hybridized to the target nucleic acid molecule; (2) a first nicking enzyme that is capable of nicking at the nicking site of said first oligonucleotide, and does not nick within said target sequence; (3) a second nicking enzyme that is capable of nicking at the nicking site of said second oligonucleotide and does not nick within said target sequence; and (4) a DNA polymerase; and   b) amplifying the double-stranded nucleic acid target sequence, wherein said amplifying comprises maintaining a reaction temperature between 54° and 60° Celsius plus or minus two degrees Celsius; wherein amplification is performed by multiple cycles of said polymerase extending said forward and reverse oligonucleotides along said target sequence producing a double-stranded nicking site, and said nicking enzymes nicking at said nicking sites, and, following a first round of amplification, at amplified copies of said sites, producing an amplification product.   
     
     
         68 . The method of  claim 67 , wherein said DNA polymerase is a thermophilic polymerase. 
     
     
         69 . The method of  claim 67 , wherein said polymerase is selected from the group consisting of Bst (large fragment), and exo-DNA Polymerase. 
     
     
         70 . The method of  claim 67 , wherein said polymerase is Bst (large fragment). 
     
     
         71 . The method of  claim 67 , wherein said nicking enzymes nick downstream of the nicking enzyme binding site. 
     
     
         72 . The method of  claim 67 , wherein said forward and reverse oligonucleotides comprise nicking enzyme binding sites recognized by the same nicking enzyme and said first and said second nicking enzymes are the same. 
     
     
         73 . The method of  claim 67 , wherein said nicking enzymes are selected from the group consisting of Nt.BspQI, Nb.BbvCi, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Bpu10I, and Nt.Bpu10I. 
     
     
         74 . The method of  claim 67 , wherein said target sequence comprises 1 nucleotide more than the sum of the nucleotides of said forward oligonucleotide recognition region and said reverse oligonucleotide recognition region. 
     
     
         75 . The method of  claim 67 , wherein said target sequence comprises 2 nucleotides more than the sum of the nucleotides of said forward oligonucleotide recognition region and said reverse oligonucleotide recognition region. 
     
     
         76 . The method of  claim 67 , wherein the target DNA molecule is selected from the group consisting of genomic DNA, plasmid, mitochondrial, and viral DNA. 
     
     
         77 . The method of  claim 67 , wherein the forward oligonucleotide is provided at the same concentration as the reverse oligonucleotide. 
     
     
         78 . The method of  claim 67 , wherein the amplification reaction is conducted for 1 to 10 minutes. 
     
     
         79 . The method of  claim 67 , further comprising detecting the amplification product. 
     
     
         80 . The method of  claim 67 , wherein said amplification product is detected by a method selected from the group consisting of gel electrophoresis, mass spectrometry, intercalating dye detection, FRET, molecular beacon detection, surface capture, capillary electrophoresis, incorporation of labeled nucleotides to allow detection by capture, fluorescence polarization, and lateral flow capture. 
     
     
         81 . The method of  claim 67 , wherein the method comprises amplifying at least two target sequences. 
     
     
         82 . The method of  claim 67 , wherein said contacting does not comprise, prior to the contacting, subjecting the target DNA molecule to a thermal denaturation step associated with amplification of the target sequence. 
     
     
         83 . The method of  claim 67 , wherein said amplifying step does not comprise temperature cycling. 
     
     
         84 . A method for amplifying a double-stranded nucleic acid target sequence, comprising:
 a) contacting a target DNA molecule comprising a double-stranded target sequence having a sense strand and an antisense strand with: (1) a first oligonucleotide and a second oligonucleotide, wherein i) said first oligonucleotide comprises a nucleic acid sequence comprising a recognition region at its 3′ end that is complementary to the target sequence antisense strand; a first nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site, wherein the portion of the nucleic acid sequence that is complementary to the target sequence strand is 8-15 nucleotides in length; wherein when the recognition region of the forward oligonucleotide is hybridized to the target sequence, the stabilizing region of the forward oligonucleotide is not hybridized to the target nucleic acid molecule; ii) said second oligonucleotide comprises a nucleotide sequence comprising a recognition region at its 3′ end that is complementary to the target sequence sense strand; a nicking enzyme binding site and a second nicking site upstream of said recognition region, and a stabilizing region upstream of said nicking site, wherein the portion of the nucleic acid sequence that is complementary to the target sequence strand is 8-15 nucleotides in length; wherein when the recognition region of the reverse oligonucleotide is hybridized to the target sequence, the stabilizing region of the reverse oligonucleotide is not hybridized to the target nucleic acid molecule; (2) a first nicking enzyme that is capable of nicking at the nicking site of said first oligonucleotide, and does not nick within said target sequence; (3) a second nicking enzyme that is capable of nicking at the nicking site of said second oligonucleotide and does not nick within said target sequence; and (4) a DNA polymerase; and   b) amplifying the double-stranded nucleic acid target sequence, wherein said amplifying comprises maintaining a reaction temperature in a narrow temperature range; wherein amplification is performed by multiple cycles of said polymerase extending said forward and reverse oligonucleotides along said target sequence producing a double-stranded nicking site, and said nicking enzymes nicking at said nicking sites, and, following a first round of amplification, at amplified copies of said sites, producing an amplification product.   
     
     
         85 . The method of  claim 84 , wherein said narrow temperature range is a few degrees Celsius. 
     
     
         86 . The method of  claim 84 , wherein said reaction temperature is between 54-60 degrees Celsius plus or minus two degrees Celsius. 
     
     
         87 . A method for amplifying at least two double-stranded nucleic acid target sequences, comprising:
 a) contacting a target DNA molecule comprising at least two double-stranded target sequences having a sense strand and an antisense strand with: (1) at least two pairs of first oligonucleotides and second oligonucleotides, wherein i) said first oligonucleotides each comprise a nucleic acid sequence comprising a recognition region at its 3′ end that is complementary to the target sequence antisense strand; a first nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site, wherein the portion of the nucleic acid sequence that is complementary to the target sequence strand is 8-15 nucleotides in length; wherein when the recognition region of the forward oligonucleotide is hybridized to the target sequence, the stabilizing region of the forward oligonucleotide is not hybridized to the target nucleic acid molecule; ii) said second oligonucleotides each comprise a nucleotide sequence comprising a recognition region at its 3′ end that is complementary to the target sequence sense strand; a nicking enzyme binding site and a second nicking site upstream of said recognition region, and a stabilizing region upstream of said nicking site, wherein the portion of the nucleic acid sequence that is complementary to the target sequence strand is 8-15 nucleotides in length; wherein when the recognition region of the reverse oligonucleotide is hybridized to the target sequence, the stabilizing region of the reverse oligonucleotide is not hybridized to the target nucleic acid molecule; (2) a first nicking enzyme that is capable of nicking at the nicking site of said first oligonucleotides, and does not nick within said target sequence; (3) a second nicking enzyme that is capable of nicking at the nicking site of said second oligonucleotides and does not nick within said target sequence; and (4) a DNA polymerase; and   b) amplifying the at least two double-stranded nucleic acid target sequences, wherein said amplifying comprises maintaining a reaction temperature between 54° and 60° Celsius plus or minus two degrees Celsius; wherein amplification is performed by multiple cycles of said polymerase extending said forward and reverse oligonucleotides along said target sequence producing a double-stranded nicking site, and said nicking enzymes nicking at said nicking sites, and, following a first round of amplification, at amplified copies of said sites, producing at least two distinct amplification products.