Method of generating long nucleic acid molecules of defined sequence

• US 7,534,569 B2
• Filed: 06/29/2007
• Issued: 05/19/2009
• Est. Priority Date: 04/11/2003
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Chang, Chu-An, Chen, Shiaw-Min
• Primary Examiner(s): Kim; Young J
• Assistant Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US11/770,787
• Publication Number:
  US 20070254343A1
• Time in Patent Office: 690 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR

Abstract

A method of generating a single-stranded nucleic acid molecule comprising (a) combining in a mixture under conditions suitable for a polymerase extension reaction, (i) a polymerase, (ii) an initial polynucleotide comprising a 5′ portion and a 3′ portion, wherein the polynucleotide forms the nucleic acid molecule 5′ end; and (iii) a plurality of overlapping template oligonucleotides each having a 5′ template portion and a 3′ portion. The method further comprises (b) hybridizing the initial polynucleotide or the extension polynucleotide and one of the template oligonucleotides; (c) incubating the mixture for sufficient time to allow an extension polynucleotide to be synthesized; (d) adding a competimer that competes with the template oligonucleotide in step (b); (e) denaturing the extension polynucleotide and template oligonucleotide; and (f) repeating steps (b), (c), (d), and (e) to generate the single-stranded nucleic acid molecule, wherein the number of repeated cycles equals the number of different template oligonucleotides.

Claims

1. A method of generating a single-stranded nucleic acid molecule, the method comprising:

(a) combining in a mixture under conditions suitable for a polymerase extension reaction;

(i) a polymerase,

(ii) an initial polynucleotide comprising a 5′

portion and a 3′

portion, wherein the polynucleotide forms the nucleic acid molecule 5′

end; and

(iii) a plurality of overlapping template oligonucleotides each having a 5′

template portion and a 3′

portion, wherein the 3′

portion of each template oligonucleotide is sufficiently complementary to the 3′

portion of one of;

the initial polynucleotide or a 3′

portion of an extension polynucleotide formed by a polymerase extension reaction of the initial polynucleotide,

(b) hybridizing the initial polynucleotide or the extension polynucleotide and one of the template oligonucleotides;

(c) incubating the mixture for sufficient time to allow an extension polynucleotide to be synthesized;

(d) adding a competimer that competes with the template oligonucleotide in step (b);

(e) denaturing the extension polynucleotide and template oligonucleotide; and

(f) repeating steps (b), (c), (d), and (e) to generate the single-stranded nucleic acid molecule, wherein the number of repeated cycles equals the number of different template oligonucleotides.

2. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the competimer in step (d) is an oligonucleotide containing a 3′

blocker and a partial sequence of the template oligonucleotide in step (b).

3. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the competimer in step (d) is an oligonucleotide that does not include a 5′

portion of the sequence of a the template oligonucleotide in step (b).

4. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the competimer in step (d) competes with the template oligonucleotide in step (b) but does not compete with a subsequent template oligonucleotide capable of hybridizing to the 3′

portion of the extension polynucleotide synthesized in step (c).

5. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the number of repeated cycles in step (f) equals the number of different template oligonucleotides.

6. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the initial polynucleotide and each of the plurality of overlapping template oligonucleotides consist of not more than 100 contiguous nucleotides.

7. The method of generating a single-stranded nucleic acid molecule according to claim 1, further comprising isolating the nucleic acid molecule from the mixture.

8. The method of generating a single-stranded nucleic acid molecule according to claim 7, wherein isolating the single-stranded nucleic acid molecule from the mixture is by binding the single-stranded nucleic acid to a column and eluting the single-stranded nucleic acid from the column.

9. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the single-stranded nucleic acid molecule contains at least about 200 bases.

10. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the single-stranded nucleic acid molecule contains at least about 500 bases.

11. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the initial polynucleotide comprises at least 20 contiguous nucleotides.

12. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the one or more template oligonucleotides comprises at least 20 contiguous nucleotides.

13. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the 3′

portion of each template oligonucleotide contains a sequence of at least 8 contiguous nucleotides which are complementary to a sequence of the same length of the 3′

portion of the initial polynucleotide or of a 3′

portion of an extension polynucleotide.

14. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the 3′

portion of each template oligonucleotide contains a sequence of at least 15 contiguous nucleotides which are complementary to a sequence of the same length of the 3′

portion of the initial polynucleotide or of a 3′

portion of an extension polynucleotide.

15. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the 3′

portion of each template oligonucleotide contains a sequence of at least 25 contiguous nucleotides which are complementary to a sequence of the same length of the 3′

portion of the initial polynucleotide or of a 3′

portion of an extension polynucleotide.

16. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the number of repeated cycles in step (e) equals at least 10 cycles.

17. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the number of repeated cycles in step (e) equals at least 20 cycles.

18. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein at least one of the plurality of overlapping template oligonucleotides comprises a 3′

blocker.

19. The method of generating a single-stranded nucleic acid molecule according to claim 18, wherein the 3′

blocker is a 3′

, amino-substituted nucleotide or a dideoxy nucleotide.

20. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein at least one of the initial oligonucleotide, template oligonucleotide, extension polynucleotide, or the single-stranded nucleic acid molecule is attached to a solid phase support.

21. The method of generating a single-stranded nucleic acid molecule according to claim 20, wherein the initial polynucleotide is attached to the solid support.

22. The method of generating a single-stranded nucleic acid molecule according to claim 20, wherein the solid phase support is selected from a polymer matrix and a controlled-pore glass.

23. The method of generating a single-stranded nucleic acid molecule according to claim 20, wherein the at least one of the initial polynucleotide or template oligonucleotide is attached at a 5′

end.

24. The method of generating a single-stranded nucleic acid molecule according to claim 20, wherein the template oligonucleotide is attached at a 3′

end.

25. The method of generating a single-stranded nucleic acid molecule according to claim 20, wherein the at least one of the initial oligonucleotide, template oligonucleotide, extension polynucleotide, or single-stranded nucleic acid molecule attached to a solid phase support is attached through a linker moiety.

26. The method of generating a single-stranded nucleic acid molecule according to claim 25, wherein the linker moiety is selected from a phosphate linker and an amine linker.

27. The method of generating a single-stranded nucleic acid molecule according to claim 1, further comprising amplifying the single-stranded nucleic acid molecule by the polymerase chain reaction.

54. The method of generating a single-stranded nucleic acid molecule according to claim 1, wherein the concentration of each subsequent template oligonucleotide is greater than the concentration of the previous template nucleotide used to form the extension polynucleotide.

28. A method of generating a single-stranded nucleic acid molecule, the method comprising:

(a) combining in a mixture under conditions suitable for a polymerase extension reaction;

(i) a polymerase,

(ii) an initial polynucleotide comprising a 5′

portion and a 3′

portion, wherein the polynucleotide forms the nucleic acid molecule 5′

end; and

(iii) a plurality of overlapping template oligonucleotides each having a 5′

template portion and a 3′

portion, wherein the 3′

portion of each template oligonucleotide is sufficiently complementary to the 3′

portion of one of;

the initial polynucleotide or a 3′

portion of an extension polynucleotide formed by a polymerase extension reaction of the initial polynucleotide,

wherein, the concentration of each subsequent template oligonucleotide is greater than the concentration of the previous template nucleotide used to form the extension polynucleotide;

(b) hybridizing the initial polynucleotide or the extension polynucleotide and one of the template oligonucleotides;

(c) incubating the mixture for sufficient time to allow an extension polynucleotide to be synthesized;

(d) denaturing to fully separate the extension polynucleotide and template oligonucleotide; and

(e) repeating steps (b), (c), and (d) to generate the single-stranded nucleic acid molecule.

29. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the number of repeated cycles in step (e) equals the number of different template oligonucleotides.

30. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the molar ratio of each subsequent template oligonucleotide to the previous template nucleotide to the initial polynucleotide or the extension polynucleotide formed by a polymerase extension reaction of the initial polynucleotide is 2:

1;

1, 2;

1.2;

1, 2;

1.5;

1, 4;

1.5;

1, 6;

1.5;

1, 10;

1.5;

1, 4;

2;

1, or 10;

2;

1.

31. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the initial polynucleotide and each of the plurality of overlapping template oligonucleotides consist of not more than 100 contiguous nucleotides.

32. The method of generating a single-stranded nucleic acid molecule according to claim 28, further comprising isolating the nucleic acid molecule from the mixture.

33. The method of generating a single-stranded nucleic acid molecule according to claim 32, wherein isolating the single-stranded nucleic acid molecule from the mixture is by binding the single-stranded nucleic acid to a column and eluting the single-stranded nucleic acid from the column.

34. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the single-stranded nucleic acid molecule contains at least about 200 bases.

35. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the single-stranded nucleic acid molecule contains at least about 500 bases.

36. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the initial polynucleotide comprises at least 20 contiguous nucleotides.

37. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the one or more template oligonucleotides comprises at least 20 contiguous nucleotides.

38. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the 3′

portion of each template oligonucleotide contains a sequence of at least 8 contiguous nucleotides which are complementary to a sequence of the same length of the 3′

portion of the initial polynucleotide or of a 3′

portion of an extension polynucleotide.

39. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the 3′

portion of each template oligonucleotide contains a sequence of at least 15 contiguous nucleotides which are complementary to a sequence of the same length of the 3′

portion of the initial polynucleotide or of a 3′

portion of an extension polynucleotide.

40. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the 3′

portion of each template oligonucleotide contains a sequence of at least 25 contiguous nucleotides which are complementary to a sequence of the same length of the 3′

portion of the initial polynucleotide or of a 3′

portion of an extension polynucleotide.

41. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the number of repeated cycles in step (e) equals at least 10 cycles.

42. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein the number of repeated cycles in step (e) equals at least 20 cycles.

43. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein at least one of the plurality of overlapping template oligonucleotides comprises a 3′

blocker.

44. The method of generating a single-stranded nucleic acid molecule according to claim 43, wherein the 3′

blocker is a 3′

, amino-substituted nucleotide or a dideoxy nucleotide.

45. The method of generating a single-stranded nucleic acid molecule according to claim 28, wherein at least one of the initial oligonucleotide, template oligonucleotide, extension polynucleotide, or the single-stranded nucleic acid molecule is attached to a solid phase support.

46. The method of generating a single-stranded nucleic acid molecule according to claim 45, wherein the initial polynucleotide is attached to the solid support.

47. The method of generating a single-stranded nucleic acid molecule according to claim 45, wherein the solid phase support is selected from a polymer matrix and a controlled-pore glass.

48. The method of generating a single-stranded nucleic acid molecule according to claim 45, wherein the at least one of the initial polynucleotide or template oligonucleotide is attached at a 5′

end.

49. The method of generating a single-stranded nucleic acid molecule according to claim 45, wherein the template oligonucleotide is attached at a 3′

end.

50. The method of generating a single-stranded nucleic acid molecule according to claim 45, wherein the at least one of the initial oligonucleotide, template oligonucleotide, extension polynucleotide, or single-stranded nucleic acid molecule attached to a solid phase support is attached through a linker moiety.

51. The method of generating a single-stranded nucleic acid molecule according to claim 50, wherein the linker moiety is selected from a phosphate linker and an amine linker.

52. The method of generating a single-stranded nucleic acid molecule according to claim 28, further comprising amplifying the single-stranded nucleic acid molecule by the polymerase chain reaction.

53. The method of generating a single-stranded nucleic acid molecule according to claim 28, further comprising adding a competimer that competes with the template oligonucleotide in step (b).