Methods for genotyping hepatitis C virus
View Patent ↗The present invention relates to methods and compositions for analyzing nucleic acids. In particular, the present invention provides methods and compositions for the detection and characterization of nucleic acid sequences and sequence changes. The methods of the present invention permit the detection and/or identification of genetic polymorphism such as those associated with human disease and permit the identification of pathogens (e.g., viral and bacterial strain identification).
1. A method of characterizing an HCV nucleic acid, comprising:
a) providing:
i) a folded target comprising an HCV nucleic acid sequence, said folded target comprising first and second non-contiguous single-stranded regions separated by an intervening region comprising a double-stranded region; and
ii) at least one oligonucleotide probe complementary to at least a portion of said folded target; and
b) mixing said folded target and said at least one oligonucleotide under conditions such that at least one of said oligonucleotide probe hybridizes to said folded target to form a probe/folded target complex,
c) quantitating the amount of said probe/folded target complex.
2. The method of claim 1 , wherein said oligonucleotide probe is completely complementary to HCV nucleic acid sequences of at least two different HCV genotypes, and wherein the amount of probe/folded target complex formed in b) distinguishes the genotype of the HCV nucleic acid sequence in said folded target from at least one other HCV genotype.
3. The method of claim 1 , wherein said at least one oligonucleotide probe is a bridging oligonucleotide probe containing regions complementary each of said first and second non-contiguous single stranded regions of said folded target.
4. The method of claim 1 , wherein said probe/folded target complex comprises a moiety that permits its capture by a solid support.
5. The method of claim 4 , wherein said detecting the presence of said probe/folded target complex comprises exposing said probe/folded target complex to a solid support under conditions such that said probe/folded target complex is captured by said solid support.
6. The method of claim 1 , wherein said folded target is labeled.
7. The method of claim 1 , wherein said probe is labeled.
8. The method of claim 1 , wherein said probe is attached to a solid support.
9. The method of claim 1 , wherein said folded target is attached to a solid support.
10. The method of claim 1 , further comprising mixing said probe/folded target complex with a reactant selected from the group consisting of structure-specific nucleases, nucleic acid polymerases, and nucleic acid ligases under conditions such that said probe oligonucleotide is modified to produce a modified oligonucleotide, wherein said detecting the presence of said probe/folded target complex comprises detecting said modified oligonucleotide.
11. The method of claim 10 , wherein said reactant is a structure-specific nuclease and said modified oligonucleotide comprises a cleaved oligonucleotide.
12. The method of claim 11 , wherein said structure-specific nuclease is a 5′ nuclease.
13. The method of claim 12 , wherein said 5′ nuclease is a FEN-1 endonuclease.
14. The method of claim 10 , wherein said reactant is a polymerase, and said modified oligonucleotide comprises an extended oligonucleotide.
15. The method of claim 10 , wherein said reactant is a ligase and said modified oligonucleotide comprises a ligated oligonucleotide.
16. The method of claim 1 , wherein said at least one oligonucleotide probe comprises DNA.
17. The method of claim 1 , wherein said folded target is DNA.
18. The method of claim 1 , wherein said at least one oligonucleotide probe has fewer than 16 contiguous nucleotides complementary to said folded target.
19. The method of claim 1 wherein said at least one oligonucleotide probe has 11 or fewer contiguous nucleotides complementary to said folded target.
20. The method of claim 3 , wherein said regions of said bridging oligonucleotide complementary to said first and second non-contiguous single stranded regions of said folded target each have 8 or fewer contiguous nucleotides complementary to said target nucleic acid.
21. A method of characterizing an HCV nucleic acid, comprising:
a) providing:
i) a first folded target comprising a first HCV nucleic acid sequence comprising a first portion, said first folded target comprising first and second non-contiguous single-stranded regions separated by an intervening region comprising a double-stranded region;
ii) a second folded target comprising a second HCV nucleic acid sequence, said second folded target comprising a first portion identical in sequence to said first portion of said first folded target;
ii) an oligonucleotide probe complementary to said first portions of said first and said second folded targets;
b) contacting said first folded target with said oligonucleotide probe under conditions such that said oligonucleotide probe binds to said first folded target to form a first probe/folded target complex;
c) contacting said second folded target with said oligonucleotide probe under the conditions of step b);
d) quantitating the amount of probe/folded target complex formed in step b) and step c), wherein a difference in the amount of probe/folded target complex formed in step b) and step c) is indicative of a sequence variation between said first folded target and said second folded target.
22. The method of claim 21 , where said contacting of step b) and step c) occurs in a single mixture.
23. The method of claim 21 , wherein said oligonucleotide probe in step c) does not substantially hybridize to said second folded target.
24. The method of claim 21 , wherein the amount of probe/folded target complex formed in step c) is reduced relative the amount of probe/folded target complex formed in step b).
25. The method of claim 21 , wherein the amount of probe/folded target complex formed in step c) is increased relative the amount of probe/folded target complex formed in step b).
26. The method of claim 21 , wherein said first and second oligonucleotides comprise DNA.