Nucleic acid accessible hybridization sites
View Patent ↗The present invention relates to methods and compositions for analyzing nucleic acids, and in particular, methods and compositions for detection and characterization of nucleic acid sequences and sequence changes. The present invention also provides methods and compositions for identifying oligonucleotides with desired hybridization properties to nucleic acid targets containing secondary structure.
1. A method for selecting a primer, comprising:
a) providing:
i) a target nucleic acid having at least one accessible site and at least one inaccessible site;
ii) a plurality of extension primers, each of said primers comprising a first region, wherein said first regions of said plurality of primers differ in sequence from each other, and wherein said plurality of primers comprise first regions that are complementary to different portions of said target nucleic acid; and
iii) a template-dependent nucleic acid extension agent;
b) exposing said plurality of extension primers and said extension agent to said target nucleic acid under conditions wherein primers comprising first regions that are complementary only to an inaccessible site in said target nucleic acid are not extended by said extension agent, and wherein primers comprising first regions that are complementary to at least one accessible site of said target nucleic acid form an extension product;
c) selecting a primer complementary to at least one accessible site by identifying a member of said plurality of primers that forms an extension product.
2. The method of claim 1 , wherein said target nucleic acid comprises DNA.
3. The method of claim 1 , wherein said target nucleic acid comprises RNA.
4. The method of claim 1 , wherein said plurality of primers further comprise a second region, said second region located 5′ of said first region.
5. The method of claim 4 , wherein said second regions of said plurality of primers are identical in sequence to one another.
6. The method of claim 5 , further comprising providing:
i) first and second amplification primers, said first amplification primer complementary to at least a portion of said second regions of said plurality of extension primers and said second amplification primer capable of hybridizing to a sequence complementary to a first domain of said target nucleic acid; and
ii) an amplification agent;
and further comprising the step of treating said extension products with said first and second amplification primers and said amplification agents to produce amplification products prior to said selecting step.
7. The method of claim 1 , wherein said plurality of primers comprises at least 10 different primers.
8. The method of claim 1 , wherein said plurality of primers comprises at least 100 different primers.
9. The method of claim 1 , wherein said plurality of primers comprises at least 1000 different primers.
10. The method of claim 1 , wherein said plurality of primers comprises a sufficient number of primers to encompass every sequence variation within said first region.
11. The method of claim 1 , wherein said first region is six or more nucleotides in length.
12. The method of claim 11 , wherein said first region is six nucleotides in length.
13. The method of claim 1 , wherein said template-dependent nucleic acid extension agent comprises a polymerase.
14. The method of claim 1 , wherein said template-dependent nucleic acid extension agent comprises a reverse transcriptase.
15. A method for identifying accessible sites on a target nucleic acid comprising:
a) providing:
i) a target nucleic acid having at least one accessible site and at least one inaccessible site;
ii) a plurality of extension primers, each of said primers comprising a first region, wherein said first regions of said plurality of primers differ in sequence from each other, and wherein said plurality of primers comprise first regions that are complementary to different portions of said target nucleic acid; and
iii) a template-dependent nucleic acid extension agent;
b) exposing said plurality of extension primers and said extension agent to said target nucleic acid under conditions wherein primers comprising first regions that are complementary only to an inaccessible site in said target nucleic acid are not extended by said extension agent, and wherein primers comprising first regions that are complementary to at least one accessible site of said target nucleic acid form an extension product that is complementary to said target nucleic acid adjacent to said accessible site;
c) determining at least a portion of the sequence of an extension product; and
d) identifying said accessible site by locating a region of said target nucleic acid adjacent to sequence that is complementary to said extension product.
16. The method of claim 15 , wherein said target nucleic acid comprises DNA.
17. The method of claim 15 , wherein said target nucleic acid comprises RNA.
18. The method of claim 15 , wherein said plurality of primers comprises at least 10 different primers.
19. The method of claim 15 , wherein said plurality of primers comprises at least 100 different primers.
20. The method of claim 15 , wherein said plurality of primers comprises at least 1000 different primers.
21. The method of claim 15 , wherein said plurality of primers comprises a sufficient number of primers to encompass every sequence variation within said first region.
22. The method of claim 15 , wherein said first region is six or more nucleotides in length.
23. The method of claim 22 , wherein said first region is six nucleotides in length.
24. The method of claim 15 , wherein said template-dependent nucleic acid extension agent comprises a polymerase.
25. The method of claim 15 , wherein said template-dependent nucleic acid extension agent comprises a reverse transcriptase.
26. A method of locating accessible sites on a target nucleic acid comprising:
a) providing:
i) a target nucleic acid having at least one accessible site and at least one inaccessible site wherein said accessible site is located a set distance from a first domain of said target nucleic acid;
ii) a plurality of extension primers, each of said primers comprising first region and second regions, wherein said first regions of said plurality of primers differ in sequence from each other, wherein said plurality of primers comprise first regions that are complementary to different portions of said target nucleic acid, and wherein said second region is located 5′ of said first region;
iii) a template-dependent nucleic acid extension agent;
iv) an amplification agent; and
v) first and second amplification primers, said first amplification primer complementary to at least a portion of said second regions of said plurality of extension primers and said second amplification primer capable of hybridizing to a sequence complementary to a first domain of said target nucleic acid;
b) exposing said plurality of extension primers and said extension agent to said target nucleic acid under conditions wherein primers comprising first regions that are complementary only to an inaccessible site in said target nucleic acid are not extended by said extension agent, and wherein primers comprising first regions that are complementary to at least one accessible site of said target nucleic acid form an extension product;
c) treating said extension products with said amplification agent and said first and second amplification primers to generate one or more amplification products, said amplification products having a length, wherein said length of said amplification products is equal to said distance of said accessible site on said target nucleic acid from said first domain of said target nucleic acid; and
d) determining a location of one or more accessible sites on said target nucleic acid using said distance.
27. The method of claim 26 , wherein said using said distance comprises determining said size of one or more of said amplification products.
28. The method of claim 26 , wherein said target nucleic acid comprises DNA.
29. The method of claim 26 , wherein said target nucleic acid comprises RNA.
30. The method of claim 26 , wherein said plurality of primers comprises at least 10 different primers.
31. The method of claim 26 , wherein said plurality of primers comprises at least 100 different primers.
32. The method of claim 26 , wherein said plurality of primers comprises at least 1000 different primers.
33. The method of claim 26 , wherein said plurality of primers comprises a sufficient number of primers to encompass every sequence variation within said first region.
34. The method of claim 26 , wherein said first region is six or more nucleotides in length.
35. The method of claim 34 , wherein said first region is six nucleotides in length.
36. The method of claim 26 , wherein said template-dependent nucleic acid extension agent comprises a polymerase.
37. The method of claim 26 , wherein said template-dependent nucleic acid extension agent comprises a reverse transcriptase.
38. The method of claim 26 , wherein said amplification agent comprises a polymerase.
39. The method of claim 38 , wherein said polymerase comprises a thermostable polymerase.
40. The method of claim 26 , wherein said treating said extension products with said amplification agent and said first and second amplification primers comprises a polymerase chain reaction.