Snap-back primers and detectable hairpin structures
View Patent ↗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.
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, and
ii) 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, and
b) 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.
2. The method of claim 1 , wherein said 3′ hairpin structure comprises a first nucleotide located immediately 5′ of said non-adjacent portion, wherein said first nucleotide corresponds to a second nucleotide at a polymorphic position in said target nucleic acid.
3. The method of claim 2 , wherein said target nucleic acid is less than 500 bases in length, and wherein said polymorphic position in said target nucleic acid is located less than 15 bases from the 3′ end of said target nucleic acid.
4. The method of claim 2 , comprising detecting the identity of said first nucleotide, thereby detecting the identity of said second nucleotide at said polymorphic position in said target nucleic acid.
5. The method of claim 4 , wherein said identity of said first nucleotide is detected using a downstream probe comprising a 3′ region configured to hybridize to said 3′ hairpin structure and a 5′ region configured to not hybridize to said 3′ hairpin structure.
6. The method of claim 5 , wherein said downstream probe hybridizes to said 3′ hairpin structure immediately 5′ of said non-adjacent portion thereby forming an invasive cleavage structure.
7. The method of claim 6 , wherein said invasive cleavage structure is formed at a temperature between 70 degrees Celsius and 95 degrees Celsius.
8. The method of claim 1 , wherein said 3′ region of said snap-back primer comprises a sequence capable of hybridizing to said non-adjacent portion of said second amplification product.
9. The method of claim 1 , wherein said 3′ region of said snap-back primer is not capable of hybridizing to said non-adjacent portion of said second amplification product.
10. The method of claim 1 , wherein said 3′ terminal portion of said 3′ hairpin structure is less than 10 nucleotides in length.
11. The method of claim 1 , wherein said 3′ snap-back portion is partially complementary to said non-adjacent portion.
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, and
b) 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.
13. The method of claim 12 , wherein the 3′ terminal nucleotide of said non-adjacent portion of said amplification product corresponds to, and is complementary to, a target nucleotide at a polymorphic position in said target nucleic acid.
14. The method of claim 13 , wherein said target nucleic acid is less than 500 bases in length, and wherein said polymorphic position in said target nucleic acid is located less than 15 bases from the 3′ end of said target nucleic acid.
15. The method of claim 13 , comprising detecting the identity of said 3′ terminal nucleotide of said non-adjacent portion, thereby detecting the identity of said target nucleotide at said polymorphic position in said target nucleic acid.
16. The method of claim 13 , wherein said identity of said 3′ terminal nucleotide is detected using an upstream probe comprising a 5′ region configured to hybridize to said amplification product and a 3′ region configured to not hybridize to said amplification product.
17. The method of claim 16 , wherein said upstream probe hybridizes to said amplification product immediately 3′ of said non-adjacent portion thereby forming an invasive cleavage structure.
18. The method of claim 17 , wherein said invasive cleavage structure is formed at a temperature between 70 degrees Celsius and 95 degrees Celsius.
19. The method of claim 12 , wherein said 3′ region of said snap-back primer comprises a sequence capable of hybridizing to the complement of said non-adjacent portion of said amplification product.
20. The method of claim 12 , wherein said 3′ region of said snap-back primer is not capable of hybridizing to the complement of said non-adjacent portion of said amplification product.
21. The method of claim 12 , wherein said 5′ terminal portion of said 5′ hairpin structure is between 10 and 30 bases in length.