Endonuclease-enhanced helicase-dependent amplification
First Claim
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1. A composition for amplifying a target nucleic acid sequence in a sample, comprisinga type II restriction endonuclease, a helicase, a DNA polymerase, a DNA molecule comprising the target nucleic acid sequence, and at least a first primer and a second primer, wherein the DNA molecule is bound by the type II restriction endonuclease, the helicase, the DNA polymerase and the at least first and second primers;
- wherein the sample may or may not contain DNA which comprises the target nucleic acid;
wherein the target nucleic acid is one which can hybridizes to at least the first and second primer and is amplified in the presence of the composition to generate an amplification product wherein the 5′
end of the amplification product is defined by the 5′
end of the first primer and the 3′
end of the amplification product is defined by the 5′
end of the second primer;
wherein the type II restriction endonuclease is one which does not recognize or cleave the amplification product; and
wherein the type II restriction endonuclease is one which cleaves the target DNA within 5000 bases of the target nucleic acid.
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Abstract
The invention provides methods and compositions for enhancing the speed and sensitivity of helicase-dependent amplification through the use of an endonuclease.
29 Citations
16 Claims
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1. A composition for amplifying a target nucleic acid sequence in a sample, comprising
a type II restriction endonuclease, a helicase, a DNA polymerase, a DNA molecule comprising the target nucleic acid sequence, and at least a first primer and a second primer, wherein the DNA molecule is bound by the type II restriction endonuclease, the helicase, the DNA polymerase and the at least first and second primers; -
wherein the sample may or may not contain DNA which comprises the target nucleic acid; wherein the target nucleic acid is one which can hybridizes to at least the first and second primer and is amplified in the presence of the composition to generate an amplification product wherein the 5′
end of the amplification product is defined by the 5′
end of the first primer and the 3′
end of the amplification product is defined by the 5′
end of the second primer;wherein the type II restriction endonuclease is one which does not recognize or cleave the amplification product; and wherein the type II restriction endonuclease is one which cleaves the target DNA within 5000 bases of the target nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 15)
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8. A kit for amplifying a target nucleic acid sequence in a sample, comprising
a type II restriction endonuclease, a helicase, a DNA polymerase, a DNA molecule comprising the target nucleic acid sequence, and at least a first primer and a second primer, wherein the DNA molecule is bound by the at least type II restriction endonuclease, the helicase, the DNA polymerase and the first and second primers wherein the sample may or may not contain DNA which comprises the target nucleic acid; -
wherein the target nucleic acid is one which can hybridize to at least the first and second primer and is amplified in the presence of the composition to generate an amplification product wherein the 5′
end of the amplification product is defined by the 5′
end of the first primer and the 3′
end of the amplification product is defined by the 5′
end of the second primer;wherein the type II restriction endonuclease is one which cannot recognize or cleave the amplification product; and wherein the type II restriction endonuclease is one which cleaves the target DNA within 5000 bases of the target nucleic acid. - View Dependent Claims (9, 10, 11, 12, 13, 14, 16)
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Specification
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Current AssigneeBioHelix Corp. (Quidel Corporation)
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Original AssigneeBioHelix Corp. (Quidel Corporation)
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InventorsKong, Huimin, Tong, Yanhong
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Primary Examiner(s)Mummert, Stephanie K
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Assistant Examiner(s)PRIEST, AARON A
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Application NumberUS14/197,130Publication NumberTime in Patent Office763 DaysField of SearchNoneUS Class Current1/1CPC Class CodesC12P 19/34 Polynucleotides, e.g. nucle...C12Q 1/6844 Nucleic acid amplification ...C12Q 1/686 Polymerase chain reaction [...C12Q 2521/301 EndonucleaseC12Q 2521/531 Glycosylase
