Isothermal DNA amplification
View Patent ↗Provided herein are nucleic acid synthesis methods and agents that employ an endonuclease for example, endonuclease V, to introduce a nick into a target DNA including one or more inosine, and uses a DNA polymerase to generate amplicons of the target DNA.
1. An amplicon production kit comprising:
at least one inosine-containing primer;
at least one exonuclease-deficient DNA polymerase with strand displacement activity; and
at least one nuclease, which is capable of nicking DNA at a residue 3′ to an inosine residue.
2. The amplicon production kit of claim 1 , wherein the at least one inosine-containing primer comprises multiple forward and reverse primers.
3. The amplicon production kit of claim 2 , further comprising at least one extender template.
4. The amplicon production kit of claim 1 , wherein the inosine is positioned at least 4 nucleotides from the 5′ end of the at least one inosine-containing primer.
5. The amplicon production kit of claim 1 , wherein the at least one nuclease comprises an endonuclease V.
6. The amplicon production kit of claim 1 , wherein the at least one exonuclease-deficient DNA polymerase is selected from exonuclease deficient T7 DNA polymerase, exo(−) Klenow, or combinations thereof.
7. The amplicon production kit of claim 5 , wherein the endonuclease V is selected from a protein, the sequence of which consists of SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, or conservative variants thereof.
8. The amplicon production kit of claim 1 , further comprising a dNTP mixture consisting of dTTP, dGTP, dATP, and dCTP, or analogs thereof.
9. The amplicon production kit of claim 1 , further comprising a chemical denaturant selected from glycerol, ethylene glycol, or formamide.
10. The amplicon production kit of claim 1 , further comprising a buffer is selected from Tis, HEPES, or MOPS.
11. The amplicon production kit of claim 10 , wherein the buffer includes a surfactant selected from Tween-20, NP-40, Triton-X-100, or a combination thereof.
12. The amplicon production kit of claim 1 , further comprising a divalent cation selected from Mn +2 , Mg +2 , or a combination thereof.
13. The amplicon production kit of claim 12 , wherein the divalent cation comprised MgCl 2 present in the reaction mixture at a final concentration of 2 mM to 6 mM.
14. The amplicon production kit of claim 1 , further comprising a reducing agent.
15. The amplicon production kit of claim 14 , wherein the reducing agent is selected from dithiothreitol (DTT), 2-mercaptoethanol (βME),2-mercaptoethylamine (MEA), or Tris(carboxyethyl) phosphine (TCEP).
16. The amplicon production kit of claim 1 , further comprising at least one single stranded DNA binding protein.
17. The amplicon production kit of claim 16 , wherein the at least one single stranded DNA binding protein is selected from E. coli SSB, T4 gene 32 protein, T7 gene 2.5 protein, Ncp7, recA, or combinations thereof.
18. The amplicon production kit of claim 1 , further comprising at least one blocking agent including albumin.
19. The amplicon production kit of claim 1 , further comprising at least one topoisomerase.
20. The amplicon production kit of claim 1 , wherein the at least one inosine-containing primer is 5 to 100 nucleotides in length, 5 to 30 nucleotides in length, or 5 to 20 nucleotides in length.
21. The amplicon production kit of claim 1 , wherein the at least one inosine-containing primer demonstrates a melting temperature of 25° C. to 70° C., 30° C. to 65° C., or 40° C. to 55° C.
22. The kit of claim 1 , wherein the at least one nuclease is incapable of degrading the at least one inosine-containing primer.