Nicking and extension amplification reaction for the exponential amplification of nucleic acids
View Patent ↗The invention is in general directed to the rapid exponential amplification of short DNA or RNA sequences at a constant temperature.
1. 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, without, prior to the contacting, subjecting the target DNA molecule to a thermal denaturation step associated with amplification of the target sequence, with:
(1) a forward template and a reverse template, wherein
i) said forward template comprises a nucleic acid sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of the target sequence antisense strand; a 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 3′ end of the target antisense strand is 8-15 nucleotides in length; wherein when the recognition region of the forward template is hybridized to the target sequence, the stabilizing region of the forward template is not hybridized to the target nucleic acid molecule;
ii) said reverse template comprises a nucleotide sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end 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, wherein the portion of the nucleic acid sequence that is complementary to the 3′ end of the target sense strand is 8-15 nucleotides in length; wherein when the recognition region of the reverse template is hybridized to the target sequence, the stabilizing region of the reverse template 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 forward template, and does not nick within said target sequence;
(3) a second nicking enzyme that is capable of nicking at the nicking site of said reverse template and does not nick within said target sequence;
(4) a DNA polymerase; and
b) amplifying the double-stranded nucleic acid target sequence under essentially isothermal conditions; wherein amplification is performed by multiple cycles of said polymerase extending said forward and reverse templates 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.
2. The method of claim 1 , wherein said DNA polymerase is a thermophilic polymerase.
3. The method of claim 1 , wherein said polymerase is selected from the group consisting of Bst (large fragment), and exo-DNA Polymerase.
4. The method of claim 1 , wherein said polymerase is Bst (large fragment).
5. The method of claim 1 , wherein said nicking enzymes nick downstream of the nicking enzyme binding site.
6. The method of claim 1 , wherein said forward and reverse templates comprise nicking enzyme binding sites recognized by the same nicking enzyme and said first and said second nicking enzymes are the same.
7. The method of claim 1 , 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.
8. The method of claim 1 , wherein said target sequence comprises 1 nucleotide more than the sum of the nucleotides of said forward template recognition region and said reverse template recognition region.
9. The method of claim 1 , wherein said target sequence comprises 2 nucleotides more than the sum of the nucleotides of said forward template recognition region and said reverse template recognition region.
10. The method of claim 1 , wherein the target DNA molecule is selected from the group consisting of genomic DNA, plasmid, mitochondrial, and viral DNA.
11. The method of claim 1 , wherein the forward template is provided at the same concentration as the reverse template.
12. The method of claim 1 , wherein the amplification is conducted between 54° C. and 60° C.
13. The method of claim 1 , wherein the amplification reaction is held at a constant temperature for 1 to 10 minutes.
14. The method of claim 1 , further comprising detecting the amplification product.
15. The method of claim 1 , 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.
16. The method of claim 1 , wherein the method comprises amplifying at least two target sequences.