Helicase dependent isothermal amplification using nicking enzymes
First Claim
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1. A method for amplifying a template nucleic acid, comprising:
- (a) providing a template nucleic acid,(b) contacting the template nucleic acid with a helicase,(c) hybridizing an oligonucleotide primer to the template nucleic acid, and(d) amplifying said template nucleic acid using the helicase dependent amplification (HDA) reaction, said HDA reaction introducing a recognition sequence into the template nucleic acid,said HDA reaction occurring in the presence of a nicking endonuclease that recognizes a nucleotide sequence of a completely or partially double-stranded nucleic acid molecule and cleaves only one strand of the template nucleic acid at a specific location relative to the recognition sequence,wherein the HDA reaction is a thermophilic HDA (tHDA),and wherein the template nucleic acid is shorter than 400 bp.
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Abstract
The present invention relates to a method for amplifying a template nucleic acid, wherein the method comprises amplifying said template nucleic acid using the helicase dependent amplification (HDA) reaction in the presence of a nicking endonuclease, and wherein said template nucleic acid comprises a sequence recognized by said nicking endonuclease or a sequence recognized by said nicking endonuclease is introduced into the template nucleic acid during the HDA reaction. The invention further pertains to a kit for amplifying a nucleic acid, comprising a nicking endonuclease, a helicase and a DNA polymerase.
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19 Claims
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1. A method for amplifying a template nucleic acid, comprising:
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(a) providing a template nucleic acid, (b) contacting the template nucleic acid with a helicase, (c) hybridizing an oligonucleotide primer to the template nucleic acid, and (d) amplifying said template nucleic acid using the helicase dependent amplification (HDA) reaction, said HDA reaction introducing a recognition sequence into the template nucleic acid, said HDA reaction occurring in the presence of a nicking endonuclease that recognizes a nucleotide sequence of a completely or partially double-stranded nucleic acid molecule and cleaves only one strand of the template nucleic acid at a specific location relative to the recognition sequence, wherein the HDA reaction is a thermophilic HDA (tHDA), and wherein the template nucleic acid is shorter than 400 bp. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method for amplifying a template nucleic acid, comprising:
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(a) providing a template nucleic acid, (b) contacting the template nucleic acid with a helicase, and (c) amplifying said template nucleic acid using the helicase dependent amplification (HDA) reaction, the template nucleic acid comprising a recognition sequence, said HDA reaction occurring in the presence of a nicking endonuclease that recognizes a nucleotide sequence of a completely or partially double-stranded nucleic acid molecule and cleaves only one strand of the template nucleic acid at a specific location relative to the recognition sequence, wherein the HDA reaction is a thermophilic HDA (tHDA), and wherein the template nucleic acid is shorter than 400 bp. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19)
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