Isothermal amplification under low salt condition
Provided herein are methods and kits for isothermal nucleic acid amplifications that use a target nucleic acid template; a reaction mixture comprising a DNA polymerase having a strand displacement activity, a deoxyribonucleoside triphosphate (dNTP) mixture, a primer with a 3′ end and a 5′ end, a molecular crowding reagent, and a buffer solution for amplifying the target nucleic acid template. The buffer solution maintains a low salt concentration of the reaction mixture, and wherein the salt concentration results in a melting temperature (T m ) of the primer at least 10° C. below the reaction temperature. The amplification is effected under isothermal condition.
1. A kit for amplifying a nucleic acid comprising:
(a) a DNA polymerase having a strand displacement activity;
(b) a molecular crowding reagent, wherein the molecular crowding reagent is a polyethylene glycol (PEG); and
(c) a buffer solution that maintains a final salt concentration between 10 mM to 20 mM during amplification at a constant reaction temperature;
further comprising a random primer;
wherein the random primer is a random hexamer primer.
2. A kit for isothermal amplification of a nucleic acid comprising:
(a) a DNA polymerase having a strand displacement activity;
(b) a molecular crowding reagent; and
(c) a buffer solution that maintains a salt concentration between 1 mM and 35 mM in an isothermal amplification reaction mixture,
wherein the molecular crowding reagent is a polyethylene glycol (PEG);
further comprising a random primer;
wherein the random primer is a random hexamer primer.
3. The kit of claim 2 , wherein the buffer provides a salt concentration between 10 mM and 30 mM in the isothermal amplification reaction mixture.
4. The kit of claim 2 , wherein the buffer provides a salt concentration between 10 mM and 20 mM in the isothermal amplification reaction mixture.
5. The kit of claim 2 , wherein the buffer provides a salt concentration of about 20 mM in the isothermal amplification reaction mixture.
6. The kit of claim 2 , wherein the PEG is selected from a group consisting of PEG-400, PEG-2000, PEG-6000, PEG-8000, and combinations thereof.
7. The kit of claim 6 , wherein the PEG is PEG-8000.
8. The kit of claim 7 , wherein a concentration of the PEG-8000 in the isothermal amplification reaction mixture is about 2.5 wt %.
9. The kit of claim 8 , wherein the buffer provides a salt concentration of 20 mM in the isothermal amplification reaction mixture.
10. The kit of claim 1 , wherein the random hexamer primer has a general structure of (atN)(atN)(atN)(atN)(atN)*N, wherein (atN) represents any of 2-amino-deoxyadenosine (2-amino-dA), dC, dG, or 2-thio-deoxythymidine (2-thio-dT), and wherein *N represents a phosphorothioate-modified random nucleotide.
11. The kit of claim 1 , wherein the random hexamer primer comprises at least one of a 2-amino-deoxyadenosine (2-amino-dA) or a 2-thio-deoxythymidine (2-thio-dT).
12. The kit of claim 2 , wherein the DNA polymerase is a phi29 DNA polymerase.
13. The kit of claim 12 further comprising a random primer having a melting temperature of lower than 20° C. in the isothermal amplification reaction mixture.
14. The kit of claim 2 further comprising deoxyribonucleoside triphosphate (dNTP) mixture.
15. A kit for isothermal amplification of a nucleic acid comprising:
(a) a phi29 DNA polymerase;
(b) a molecular crowding reagent;
(c) a buffer solution that maintains a salt concentration of about 20 mM in an isothermal amplification reaction mixture; and
(d) a random hexamer primer having an oligonucleotide sequence NNNN*N*N, wherein the molecular crowding reagent is polyethylene glycol-8000 (PEG-8000), and wherein *N represents a phosphorothioate-modified random nucleotide.
16. The kit of claim 15 , wherein a melting temperature of the random hexamer primer in the isothermal amplification reaction mixture is lower than 20° C.
17. The kit of claim 2 , wherein the random hexamer primer has a general structure of (atN)(atN)(atN)(atN)(atN)*N, wherein (atN) represents any of 2-amino-deoxyadenosine (2-amino-dA), dC, dG, or 2-thio-deoxythymidine (2-thio-dT), and wherein *N represents a phosphorothioate-modified random nucleotide.
18. The kit of claim 2 , wherein the random hexamer primer comprises at least one of a 2-amino-deoxyadenosine (2-amino-dA) or a 2-thio-deoxythymidine (2-thio-dT).