Isothermal amplification under low salt condition
First Claim
Patent Images
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 (Tm) of the primer at least 10°
C. below the reaction temperature, andwherein the molecular crowding reagent is polyethylene glycol (PEG).
3 Assignments
0 Petitions
Accused Products
Abstract
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 (Tm) of the primer at least 10° C. below the reaction temperature. The amplification is effected under isothermal condition.
12 Citations
23 Claims
-
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; andc) amplifying the target nucleic acid template under isothermal amplification conditions at a constant reaction temperature, wherein the salt concentration results in a melting temperature (Tm) of the primer at least 10°
C. below the reaction temperature, andwherein the molecular crowding reagent is polyethylene glycol (PEG). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
-
-
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; andc) 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 (Tm) of the random hexamer primer at least 10°
C. below the reaction temperature.
-
Specification