Rolling circle synthesis of oligonucleotides and amplification of select randomized circular oligonucleotides

• US 5,714,320 A
• Filed: 02/23/1995
• Issued: 02/03/1998
• Est. Priority Date: 04/15/1993
• Status: Expired due to Term

• Current Assignee: University of Rochester
• Original Assignee: University of Rochester
• Inventors: Kool, Eric T.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/393,439
• Time in Patent Office: 1,076 Days
• Field of Search: 435/6, 435/5, 435/91.1, 435/91.2, 514/44, 536/24.5, 536/24.3-24.33, 536/23.1
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/337 Ribozyme
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2537/162 Helper probe

Abstract

The present invention provides methods for synthesis, selection, and amplification of DNA and RNA oligonucleotides and analogs. The method for synthesizing an oligonucleotide involves: providing an effective amount of an isolated circular oligonucleotide template which comprises at least one copy of the desired oligonucleotide sequence linked to a cleavage site; providing an effective amount of an isolated oligonucleotide primer; annealing the primer to the circular template to form a primed circular template; and combining the primed circular template with an effective amount of at least two types of nucleotide triphosphates and an effective amount of a polymerase enzyme to form a nucleotide multimer complementary to the circular oligonucleotide template, wherein the nucleotide multimer comprises multiple copies of the oligonucleotide sequence joined end to end. Preferably, the nucleotide multimer is cleaved to produce oligonucleotides having well-defined ends.

Claims

1. A method for synthesizing a selected oligonucleotide having well-defined ends comprising:

(a) annealing an effective amount of an oligonucleotide primer to a single-stranded circular template to yield a primed circular template, wherein the single-stranded circular template comprises (i) at least one copy of a nucleotide sequence complementary to the sequence of the selected oligonucleotide and (ii) at least one nucleotide effective to produce a cleavage site in the oligonucleotide multimer;

(b) combining the primed circular template with an effective amount of at least two types of nucleotide triphosphates and an effective amount of a polymerase enzyme without the addition of auxiliary proteins to yield a single-stranded oligonucleotide multimer complementary to the circular oligonucleotide template, wherein the oligonucleotide multimer comprises multiple copies of the selected oligonucleotide; and

(c) cleaving the oligonucleotide multimer at the cleavage site to produce the selected oligonucleotide having well-defined ends.

2. The method of claim 1 wherein the step of cleaving the oligonucleotide multimer occurs after the oligonucleotide multimer is completely formed.

3. The method of claim 1 wherein the step of cleaving the oligonucleotide multimer occurs as the oligonucleotide multimer is being formed.

4. The method of claim 1 wherein the step of cleaving the oligonucleotide multimer includes cleaving the oligonucleotide multimer with a restriction enzyme.

5. The method of claim 1 wherein the cleavage site comprises a cleavable natural nucleotide not present in the selected oligonucleotide sequence, and the step of cleaving the oligonucleotide multimer includes chemically cleaving the oligonucleotide multimer at the cleavable natural nucleotide.

6. The method of claim 1 wherein the cleavage site includes a cleavable modified nucleotide.

7. The method of claim 6 wherein the modified nucleotide includes a photolabile group, and the step of cleaving the oligonucleotide multimer includes cleaving the photolabile group with light.

8. The method of claim 1 wherein each of the circular oligonucleotide template, the oligonucleotide primer, and the nucleotide multimer is single-stranded.

9. The method of claim 8 wherein the single-stranded oligonucleotide primer is a DNA oligomer.

10. The method of claim 9 wherein the polymerase enzyme is selected from the group consisting of DNA Polymerase I, Klenow fragment of DNA Polymerase I, T4 DNA Polymerase, T7 DNA Polymerase, Taq Polymerase, and AMV Reverse Transcriptase, and homologs of each having at least about 80% homology to said polymerase enzyme.

11. The method of claim 10 wherein the polymerase enzyme is a Klenow fragment of DNA Polymerase I.

12. The method of claim 8 wherein the single-stranded oligonucleotide primer is an RNA oligomer.

13. The method of claim 12 wherein the polymerase enzyme is selected from the group consisting of T7 RNA Polymerase, T4 RNA Polymerase, SP6 RNA Polymerase, RNA Polymerase II, RNA Polymerase III, T3 RNA Polymerase, E. coli RNA Polymerase and homologs thereof having at least about 80% homology.

14. The method of claim 8 wherein the single-stranded oligonucleotide primer is a synthetically modified analog of DNA or RNA.

15. The method of claim 8 wherein the single-stranded oligonucleotide primer comprises about 4-50 nucleotides.

16. The method of claim 8 wherein the single-stranded oligonucleotide circular template comprises about 15-1500 nucleotides.

17. The method of claim 1 wherein the primer and circular template are provided in an effective catalytic amount.

18. The method of claim 1 wherein the primer is provided in an amount of about 0.1-100 moles per mole of circular template.

19. The method of claim 1 wherein the nucleotide triphosphates are provided in an amount of about 50-10⁷ moles per mole of circular template.

20. The method of claim 1 wherein the circular oligonucleotide template is prepared by a process comprising the steps of:

(a) hybridizing each end of a linear precursor oligonucleotide to a single positioning oligonucleotide having a 5'"'"' nucleotide sequence complementary to a portion of the sequence comprising the 3'"'"' end of the linear precursor oligonucleotide and a 3'"'"' nucleotide sequence complementary to a portion of the sequence comprising the 5'"'"' end of the linear precursor oligonucleotide, to yield an open oligonucleotide circle wherein the 5'"'"' end and the 3'"'"' end of the open circle are positioned so as to abut each other; and

(b) joining the 5'"'"' end and the 3'"'"' end of the open oligonucleotide circle to yield a circular oligonucleotide template.

21. The method of claim 1 wherein the single-stranded circular oligonucleotide template comprises about 15-1500 nucleotides.

22. The method of claim 21 wherein the single-stranded circular oligonucleotide template comprises about 24-500 nucleotides.

23. The method of claim 22 wherein the single-stranded circular oligonucleotide template comprises about 30-150 nucleotides.

24. The method of claim 1 wherein the selected oligonucleotide comprises about 4-1500 nucleotides.

25. The method of claim 1 wherein the oligonucleotide multimer is about 4-4000 times the length of a linearized circular oligonucleotide template.

26. The method of claim 1 wherein the oligonucleotide multimer has about 60 to 5,000,000 nucleotides.

27. The method of claim 26 wherein the oligonucleotide multimer has about 500-100,000 nucleotides.

28. The method of claim 1 wherein the oligonucleotide multimer has 5-100,000 copies of the selected oligonucleotide.

29. The method of claim 1 wherein the single-stranded circular template consists of the at least one copy of a nucleotide sequence complementary to the sequence of the selected oligonucleotide and the at least one nucleotide effective to produce a cleavage site on the oligonucleotide multimer.

30. The method of claim 29 wherein the single-stranded circular template consists of a single copy of a nucleotide sequence complementary to the selected oligonucleotide sequence and the at least one nucleotide effective to produce a cleavage site on the oligonucleotide multimer.

31. The method of claim 1 or 29 wherein the nucleotide sequence complementary to the sequence of the selected oligonucleotide comprises the at least one nucleotide effective to produce a cleavage site on the oligonucleotide multimer.

32. The method of claim 1 wherein the step of cleaving further includes annealing to the oligonucleotide multimer an adaptor oligonucleotide complementary to a region of the oligonucleotide multimer to produce a cleavage site on the oligonucleotide multimer cleaved by a restriction enzyme.

33. The method of claim 32 wherein the restriction enzyme is a Type II restriction enzyme, and wherein the adaptor oligonucleotide comprises a double-stranded region comprising a recognition site for the Type II restriction enzyme.

34. The method of claim 4 wherein the step of cleaving further includes annealing to the oligonucleotide multimer an adaptor oligonucleotide having a sequence complementary to a region of the oligonucleotide multimer comprising the cleavage site to yield a double-stranded site cleaved by the restriction enzyme.

35. The method of claim 34 wherein the restriction enzyme is a Type II restriction enzyme, and wherein the adaptor oligonucleotide comprises a double-stranded region comprising a recognition site for the Type II restriction enzyme.

36. The method of claim 35 wherein the double-stranded region of the adaptor oligonucleotide is formed by a DNA hairpin.

37. The method of claim 4 wherein the restriction enzyme cleaves the oligonucleotide multimer at a double-stranded site comprising the cleavage site, which double-stranded site is formed by interstrand or intrastrand base-pairing of the oligonucleotide multimer.

38. The method of claim 37 wherein the double-stranded site is a DNA hairpin formed by intrastrand base-pairing of the oligonucleotide multimer, and wherein the step of cleaving the oligonucleotide multimer includes excising the hairpins from the oligonucleotide multimer with a Type II restriction enzyme.

39. The method of claim 4 wherein the restriction enzyme cleaves the oligonucleotide multimer at a single-stranded site comprising the cleavage site.

40. The method of claim 4 wherein the selected oligonucleotide comprises at least one nucleotide effective to produce a cleavage site on the oligonucleotide multimer.

41. The method of claim 40 further comprising contacting the the selected oligonucleotide with a nuclease or restriction enzyme to remove the at least one cleavage site nucleotide.

42. The method of claim 41 wherein the nuclease used to remove the at least one cleavage site nucleotide from the selected oligonucleotide is an exonuclease.

43. The method of claim 1 wherein the step of cleaving the oligonucleotide multimer comprises cleaving the oligonucleotide multimer at the cleavage site with an endonuclease.

44. The method of claim 5 wherein the cleavable natural nucleotide occurs once between each oligonucleotide in the oligonucleotide multimer.

45. The method of claim 26 wherein the positioning oligonucleotide is the oligonucleotide primer.

46. The method of claim 1 wherein the single-stranded circular template contains no nucleotides other than i) the at least one copy of a nucleotide sequence complementary to the sequence of the selected oligonucleotide, and (ii) the at least one nucleotide effective to produce a cleavage site in the oligonucleotide multimer.

47. The method of claim 1 wherein the effective amount of the oligonucleotide primer is a catalytic quantity.