High specificity primers, amplification methods and kits
First Claim
Patent Images
1. A hairpin oligonucleotide primer for extension by a DNA polymerase 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 complementarily 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.
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Citations
25 Claims
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1. A hairpin oligonucleotide primer for extension by a DNA polymerase 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 complementarily 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, 17, 18, 19, 20, 21, 22)
- arm sequence, a 5′
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23. In a linear oligonucleotide primer for extension by a DNA polymerase when hybridized to a complementary sequence in a target strand, said primer having a 5′
- terminus and a 3′
terminal region, the improvement comprising adding to said 5′
terminus a nucleotide sequence having sufficient complementarity to said 3′
terminal region to form a hairpin structure comprising a double-stranded stem and a single-stranded loop, wherein contacting said extended primer to said target causes said stem to dissociate, wherein hybridization of the loop to a model oligonucleotide having the same length as the loop and being perfectly complementary thereto does not cause said stem to dissociate, and wherein said stem lacks a 3′
single-stranded overhang sufficient to nucleate hybridization of the primer to its target. - View Dependent Claims (24, 25)
- terminus and a 3′
Specification