Snap-back primers and detectable hairpin structures
First Claim
1. A method for forming a 3′
- hairpin structure, comprising;
a) contacting a sample suspected of containing a target nucleic acid with a snap-back primer and a reverse primer, wherein said snap-back primer comprises;
i) a 3′
region configured to hybridize to said target nucleic acid such that it can be extended by a polymerase, and ii) a 5′
region that is configured to not hybridize to said target nucleic when said 3′
region of said snap-back primer is hybridized to said target nucleic acid; and
wherein said contacting is under conditions such that;
i) said 3′
region of said snap-back primer hybridizes to said target nucleic acid and is extended to generate a first amplification product, andii) said reverse primer hybridizes to said first amplification product and is extended to generate a second amplification product, wherein said second amplification product comprises a 3′
snap-back portion capable of hybridizing to a non-adjacent portion of said second amplification product, andb) treating said sample under conditions such that said second amplification product is separated from said first amplification product, and said second amplification product forms a 3′
hairpin structure, wherein said 3′
hairpin structure comprises;
i) said 3′
snap-back portion hybridized to said non-adjacent portion; and
ii) a 3′
terminal portion not hybridized to said second amplification product.
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Abstract
The present invention provides methods, compositions, and kits comprising snap-back primers used for forming 3′ hairpin structures, 5′ hairpin structures, and double hairpin structures. The hairpin structures may be used for detecting target sequences (e.g., such as small RNA target sequence), for detecting polymorphisms in target sequences (e.g., such as polymorphisms located near the 5′ or 3′ ends of the target sequence), or other nucleic acid characterization methods. In certain embodiments, the hairpin structures form invasive cleavage structures (e.g., in combination with a probe or upstream oligonucleotide) which may be cleaved by structure-specific enzymes in order to detect the presence or absence of a particular nucleotide or nucleotide sequence.
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Citations
21 Claims
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1. A method for forming a 3′
- hairpin structure, comprising;
a) contacting a sample suspected of containing a target nucleic acid with a snap-back primer and a reverse primer, wherein said snap-back primer comprises;
i) a 3′
region configured to hybridize to said target nucleic acid such that it can be extended by a polymerase, and ii) a 5′
region that is configured to not hybridize to said target nucleic when said 3′
region of said snap-back primer is hybridized to said target nucleic acid; and
wherein said contacting is under conditions such that;i) said 3′
region of said snap-back primer hybridizes to said target nucleic acid and is extended to generate a first amplification product, andii) said reverse primer hybridizes to said first amplification product and is extended to generate a second amplification product, wherein said second amplification product comprises a 3′
snap-back portion capable of hybridizing to a non-adjacent portion of said second amplification product, andb) treating said sample under conditions such that said second amplification product is separated from said first amplification product, and said second amplification product forms a 3′
hairpin structure, wherein said 3′
hairpin structure comprises;
i) said 3′
snap-back portion hybridized to said non-adjacent portion; and
ii) a 3′
terminal portion not hybridized to said second amplification product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
- hairpin structure, comprising;
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12. A method for forming a 5′
- hairpin structure, comprising;
a) contacting a sample suspected of containing a target nucleic acid with a snap-back primer comprising;
i) a 3′
region configured to hybridize to said target nucleic acid such that it can be extended by a polymerase, and ii) a 5′
region that is configured to not hybridize to said target nucleic acid when said 3′
region of said snap-back primer is hybridized to said target nucleic acid; and
wherein said contacting is under conditions such that said 3′
region of said snap-back primer hybridizes to said target nucleic acid and is extended to generate an amplification product, wherein said amplification product comprises a 5′
snap-back portion capable of hybridizing to a non-adjacent portion of said amplification product, andb) treating said sample under conditions such that said amplification product is separated from said target nucleic acid, and said amplification product forms a 5′
hairpin structure, wherein said 5′
hairpin structure comprises;
i) said 5′
snap-back portion hybridized to said non-adjacent portion, and ii) a 5′
terminal portion that is not hybridized to said amplification product. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21)
- hairpin structure, comprising;
Specification