Isothermal amplification under low salt condition
View Patent ↗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 method for amplifying a nucleic acid, comprising:
a) providing a target nucleic acid template;
b) contacting the target nucleic acid template with 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, wherein the buffer solution maintains a salt concentration of the reaction mixture between 1 to 75 mM; and
c) amplifying the target nucleic acid template under isothermal amplification conditions at a constant reaction temperature,
wherein the salt concentration results in a melting temperature (T m ) of the primer at least 10° C. below the reaction temperature, and
wherein the molecular crowding reagent is polyethylene glycol (PEG).
2. The method of claim 1 , wherein the primer has a length between 5 nucleotides to 9 nucleotides and the salt concentration is maintained between 1 to 35 mM.
3. The method of claim 2 , wherein the salt concentration of the reaction mixture is maintained between 10 to 30 mM.
4. The method of claim 2 , wherein the salt concentration of the reaction mixture is maintained at about 20 mM.
5. The method of claim 1 , wherein an input quantity of the target nucleic acid template is at least 5 femtograms.
6. The method of claim 5 , wherein the target nucleic acid is isolated from a bacterial source.
7. The method of claim 1 , wherein the target nucleic acid is isolated a human source, and wherein an input quantity of the target nucleic acid is at least 5 picograms.
8. The method of claim 1 , wherein the PEG is selected from a group consisting of a PEG 400, PEG 2000, PEG 6000, PEG 8000and combinations thereof.
9. The method of claim 1 , wherein amplifying the nucleic acid template comprises a rolling circle amplification (RCA) or a multiple displacement amplification (MDA).
10. The method of claim 1 , wherein amplifying the nucleic acid template is performed under high stringency conditions.
11. The method of claim 1 , wherein the DNA polymerase is phi29 DNA polymerase.
12. The method of claim 1 , wherein the reaction temperature is in a range of 25 to 35° C.
13. The method of claim 1 , wherein the primer is a random primer.
14. The method of claim 1 , wherein the primer is thioated.
15. The method of claim 1 , wherein the primer comprises a nucleotide analogue.
16. The method of claim 15 , wherein the primer comprises a phosphorothioate linkage between a 3′ terminal nucleotide and a nucleotide that is adjacent to the 3′ terminal nucleotide.
17. The method of claim 15 , wherein the primer comprises a locked nucleic acid (LNA) that precedes a nucleotide base.
18. The method of claim 15 , wherein the nucleotide analogue in the primer is 2-amino-deoxyadenosine (2-amino-dA).
19. The method of claim 18 , wherein the primer further comprises a nucleotide analogue 2-thio-deoxythymidine (2-thio-dT) to prevent a primer-dimer formation.
20. The method of claim 1 , wherein the primer is a hexamer.
21. The method of claim 20 , wherein the primer has a sequence of NNNN*N*N.
22. The method of claim 20 , wherein the hexamer has a general structure of (atN)(atN)(atN)(atN)(atN)*N, wherein (atN) is the 5′ end and *N is the 3′ end nucleotide of the hexamer, and wherein “N” represents a deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosine (dG), or deoxythymidine (dT), (atN) represents a random mixture of 2-amino-dA, dC, dG, and 2-thio-dT, and “*” represents a phosphorothioate linkage.
23. A method for amplifying a nucleic acid, comprising:
a) providing a target nucleic acid template;
b) contacting the target nucleic acid template with a reaction mixture comprising a DNA polymerase having a strand displacement activity, a deoxyribonucleoside triphosphate (dNTP) mixture, a random hexamer primer with a 3′ end and a 5′ end, polyethylene glycol as a molecular crowding reagent, and a buffer solution, wherein the buffer solution maintains a salt concentration of the reaction mixture at 15 mM; and
c) amplifying the target nucleic acid template under isothermal conditions at a constant temperaure of 30° C.;
wherein the salt concentration results in a melting temperature (T m ) of the random hexamer primer at least 10° C. below the reaction temperature.