Solid phase isothermal amplification
First Claim
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1. A method for amplifying a nucleic acid, comprising:
- a) providing a target nucleic acid template on a solid support;
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.
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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.
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27 Claims
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1. A method for amplifying a nucleic acid, comprising:
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a) providing a target nucleic acid template on a solid support; 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. - 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, 24, 25, 26, 27)
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Specification