A METHOD FOR INCREASING THE AFFINITY OF AN OLIGONUCLEOTIDE FOR A TARGET NUCLEIC ACID

• US 20060099584A1
• Filed: 12/24/2002
• Published: 05/11/2006
• Est. Priority Date: 05/01/2002
• Status: Abandoned Application

• Current Assignee: Nucleics Pty Limited
• Original Assignee: Nucleics Pty Limited
• Inventors: Tillett, Daniel, Thomas, Torsten
• Application Number:
  US10/513,076
• Publication Number:
  US 20060099584A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

The present invention relates to the optimization of primer libraries. Shorter primers are annealed to template sequences and extended in order to provide primers having improved specificity. The primers of the invention have utility in DNA amplification and sequencing methods.

Claims

1. A method of increasing the affinity of an extendable oligonucleotide (EO) for a target nucleic acid comprising:

(a) hybridisation of the EO to a template oligonucleotide (TO) via a region of complementarity, wherein the 5′

region of the TO

(i) overhangs the 3′

end of the EO; and

(ii) bears homology to the target nucleic acid; and

(b) extension of the EO such that at least one nucleotide complementary to the TO is added to the 3′

end of the EO, resulting in an extended EO.

2. A method according to claim 1 wherein the EO is of equal or shorter length than the TO.

3. A method according to claim 1 or claim 2 wherein the EO and TO comprise deoxyribonucleic acids.

4. A method according to claim 1 wherein the 5′

region of the TO overhangs the 3′

end of the EO by two to six nucleic acids.

5. A method according to claim 1 wherein extension of the EO is achieved by a polymerase.

6. A method according to claim 5 wherein the polymerase is selected from the following:

E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase, Vent DNA polymerase, Vent (exo⁻

), Deep Vent, Deep vent (exo⁻

), 9.degree. N DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, T7 RNA polymerase, M-MuLV reverse transcriptase, SP6 RNA polymerase or Taq DNA polymerase.

7. A method according to claim 5 or claim 6 wherein the polymerase has no 5′

to 3′

or 3′

to 5′

exonuclease activities.

8. A method according to claim 7 wherein the polymerase is Klenow 3′

to 5′

exonuclease minus.

9. A method according to claim 1 wherein the extended EO is purified.

10. A method according to claim 1 wherein the extended EO is dissociated from the TO and used to bind to the target nucleic acid in a further method.

11. A method according to claim 10 wherein the further method is selected from the following:

polymerase chain reaction (PCR), ligation chain reaction (LCR), reverse-transcriptase PCR (T-PCR), primer extension reaction for mRNA-transcript analysis, self-sustaining sequence replication, rolling circle amplification, strand displacement amplification, isothermal DNA amplification and DNA-sequencing.

12. A method according to claim 1 wherein the 3′

end of the TO is extendable by a polymerase.

13. A method according to claim 1 wherein extension of the TO is blocked.

14. A method according to claim 13 wherein extension of the TO is blocked by a TO design that creates a non-hybridising 5′

overhang of the EO, providing no template for the extension of the TO.

15. A method according to claim 13 wherein extension of the TO is blocked by modification of the 3′

end of the TO rendering it unrecognisable or non-extendable by a polymerase.

16. A method according to claim 15 wherein the modification of the 3′

end of the TO is by addition of a phosphate group, biotin, carbon chain, amine or dideoxyribonucleotide to the 3′

end of the TO.

17. A method according to claim 1 wherein the EO and/or TO comprise a degenerate or universal nucleotide.

18. A method according to claim 17 wherein the universal nucleotide is selected from the following:

inosine, 3-nitropyrrole and 5-nitroindole.

24. Two complementary libraries comprising, respectively, EOs and TOs, wherein the EOs and TOs are suitable for use in a method according to any one of claims 1 to 20.

29. A method according to claim 1 wherein the TO includes a catch region comprising one or more degenerate or universal nucleotides.

30. A method according to claim 29 wherein the catch region lies between a constant 3′

region of the TO and a variable region.

32. A method according to claim 29 wherein the degenerate or universal positions of the catch region hybridise in most or all of its positions with most or all members of the EO library.

33. A method according to claim 1 wherein the nucleotides closest to the 3′

end of the EO are G or C.

34. A method according to any one of claims 1, 19 or claim 20 wherein the EO and TO comprise the following nucleotides:

35. A method according to claim 34 wherein the sequence of the TO is 3′

YYYYYNNNSSXX)X 5′

.

19. A method of amplifying a target nucleic acid comprising

(a) hybridisation of an extendable oligonucleotide (BO), to a template oligonucleotide (TO), wherein the 5′

region of the TO

(i) overhangs the EO by at least one nucleotide; and

(ii) bears homology to the target nucleic acid;

(b) extension of the EO such that at least one nucleotide complementary to the TO is added to the 3′

end of the EO; and

(c) amplification of the target nucleic acid utilising the extended EO.

20. A method of sequencing a target nucleic acid comprising

(a) hybridisation of an extendable oligonucleotide (EO) to a template oligonucleotide (TO), wherein the 5′

region of the TO

(i) overhangs the EO by at least one nucleotide; and

(ii) bears homology to the target nucleic acid; and

(b) extension of the EO such that at least one nucleotide complementary to the TO is added to the 3′

end of the EO; and

(c) dissociation of the annealed oligonucleotides and utilising the extended EO in a sequencing reaction.

21. A pair of oligonucleotides comprising an extendable oligonucleotide (EO) and a template oligonucleotide (TO) wherein

(a) the EO comprises a region complementary to a region of the TO;

(b) the EO is extendable at its 3′

end; and

(c) wherein the 5′

end of the TO is such that if the EO and TO were annealed, the 5′

end of the TO would overhang the 3′

end of the EO by at least one nucleotide.

22. A pair of oligonucleotides according to claim 21 wherein the at least one nucleotide is substantially similar to, or identical with, a nucleotide in a target nucleic acid.

23. A library comprising a plurality of pairs of oligonucleotides according to claim 22.

36. A pair of oligonucleotides according to claim 21 wherein the EO and TO comprise the following nucleotides:

37. A pair of oligonucleotides according to claim 36 wherein the sequence of the TO is 3′

YYYYYNNNSSXXXXX 5′

.

25. A kit comprising a library of extendable oligonucleotides (EOs) and a complementary library of template oligonucleotides (TOs) wherein

(a) the EOs comprise a region complementary to a region of the TOs herein called a clamp;

(b) the EO is extendable at its 3′

end; and

(c) wherein the 5′

end of the TOs is such that when an EO from the library of EOs and a TO from the library of TOs are annealed, the 5′

end of the TO overhangs the 3′

end of the EO by at least one nucleotide.

26. A kit according to claim 25 wherein the clamp comprises a sequence motif useful for subsequent applications.

27. A kit according to claim 26 wherein the sequence motif is a recognition sequence for a restriction endonuclease, a phage polymerase transcription signal, a binding site for ribosomes, or a start codon enabling translation.

28. A kit according to claim 27 wherein the clamp is a region that is fully complementary between the EO and TO.

31. A method according to claim 29 wherein the catch region is adjacent to, or forms part of, a clamp region as defined in claim 25.

38. A kit according to claim 25 wherein the EOs and TOs comprise the following nucleotides:

39. A kit according to claim 38 wherein the sequence of the TOs is 3′

YYYYYNNNSSXXXXX 5′

.