High specificity primers, amplification methods and kits
First Claim
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1. A process for nucleic acid amplification comprising the extension by a DNA polymerase of at least one hairpin oligonucleotide primer comprising a 3′
- arm sequence, a 5′
arm sequence and a single-stranded loop sequence separating said arm sequences, said 3′
arm sequence and said loop sequence both being perfectly complementary to a selected priming region of a target nucleic acid strand, and said arm sequences having sufficient complementarity to one another to form a double-stranded stem hybrid in the absence of the target strand, wherein said primer hybridizes to said target strand overcoming the stem hybrid, wherein said stem hybrid is sufficiently strong that hybridization of the loop sequence to a sequence of the length of the loop and perfectly complementary to the loop sequence does not cause dissociation of the stem, and wherein said 3′
arm sequence contains insufficient nucleotides that are not complementary to said 5′
arm sequence to nucleate hybridization of the primer to the target.
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Abstract
For nucleic acid amplification including extension of primers by a DNA polymerase, high specificity primers are provided. The primers include a type of hairpin structure in which a single-stranded loop separates complementary 3′ and 5′ arms and in which the loop and the 3′ arm are complementary to the target nucleic acid. Amplification methods, assays and kits including such primers are included in the invention.
136 Citations
16 Claims
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1. A process for nucleic acid amplification comprising the extension by a DNA polymerase of at least one hairpin oligonucleotide primer comprising a 3′
- arm sequence, a 5′
arm sequence and a single-stranded loop sequence separating said arm sequences, said 3′
arm sequence and said loop sequence both being perfectly complementary to a selected priming region of a target nucleic acid strand, and said arm sequences having sufficient complementarity to one another to form a double-stranded stem hybrid in the absence of the target strand, wherein said primer hybridizes to said target strand overcoming the stem hybrid, wherein said stem hybrid is sufficiently strong that hybridization of the loop sequence to a sequence of the length of the loop and perfectly complementary to the loop sequence does not cause dissociation of the stem, and wherein said 3′
arm sequence contains insufficient nucleotides that are not complementary to said 5′
arm sequence to nucleate hybridization of the primer to the target. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
- arm sequence, a 5′
Specification
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Current AssigneeRutgers University
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Original AssigneeThe Public Health Research Institute of the City of New York, Inc.
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InventorsKramer, Fred R., Tyagi, Sanjay, Vartikian, Robert
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Primary Examiner(s)Whisenant, Ethan
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Assistant Examiner(s)LU, FRANK WEI MIN
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Application NumberUS09/317,350Time in Patent Office820 DaysField of Search435/6, 435/91.1, 435/91.2, 435/975, 435/183, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 436/94, 436/501US Class Current435/91.2CPC Class CodesC12Q 1/6818 involving interaction of tw...C12Q 1/6844 Nucleic acid amplification ...C12Q 2525/301 Hairpin oligonucleotidesC12Q 2561/113 Real time assayC12Q 2565/101 Interaction between at leas...Y10T 436/143333 Saccharide [e.g., DNA, etc.]
