Cleavable hairpin primers
AssigneeLuminex Corporation
InventorsMartin KOCHANCZYK, Doug WHITMAN
Life of this patent
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Abstract
Nucleic acid constructs and methods that provide superior prevention of primer-dimers and other artifacts of false priming events are disclosed. In particular, there is disclosed a hairpin primer having a target-specific primer region, wherein the target-specific region comprises a target-binding dependent cleavage sequence; a first stem forming region 5′ of the target-specific primer region; and a second stem forming region 3′ of the target-specific primer region, wherein the second stem forming region is complementary to the first stem forming region. Methods of using the hairpin primer to amplify a target nucleic acid are also disclosed.
Claims18
A method of amplifying a target nucleic acid, the method comprising:
- a) Contacting a sample with a hairpin primer comprising a target-nucleic acid complementary region, a target-binding dependent cleavage sequence and complementary first and second stem-forming regions,
- b) Hybridizing the hairpin primer comprising a target nucleic acid complementary region to the target nucleic acid if present in the sample,
- c) Cleaving the hybridized hairpin primer with a cleaving agent that recognizes the target-binding dependent cleavage sequence of the hairpin primer to obtain an extendable primer;
- and
- d) Extending the primer to create an amplified target nucleic acid.
A method of detecting a single nucleotide polymorphism at a discriminating nucleotide in a target nucleic acid, the method comprising:
- a. Contacting a sample with at least a first and a second hairpin primer, each of said first and second hairpin primers comprising a target-nucleic acid complementary region, a target-binding dependent cleavage sequence and complementary first and second stem-forming regions, wherein the target-binding dependent cleavage sequence of the first hairpin primer comprises a ribonucleotide complementary to a wildtype nucleotide, and the target-binding dependent cleavage sequence of the second hairpin primer comprises a ribonucleotide complementary to a polymorphic nucleotide;
- b. Hybridizing the first and second hairpin primers to the target nucleic acid if present in the sample;
- c. Cleaving any first and second hairpin primers hybridized to the target nucleic acid with a cleavage agent that recognizes the target-binding dependent cleavage sequence of the first and second hairpin primers to create first and second extendable primers;
- d. Extending the first and second extendable primers to create first and second extension products;
- and
- e. Detecting the presence or absence of the single nucleotide polymorphism by detecting the presence or absence of first and second extension products.
Dependencies are inferred from the claim text.