Method of generating long nucleic acid molecules of defined sequence

• US 20040241732A1
• Filed: 04/07/2004
• Published: 12/02/2004
• Est. Priority Date: 04/11/2003
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Chen, Shiaw-Min, Chang, Chu-an
• Application Number:
  US10/819,656
• Publication Number:
  US 20040241732A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 of a predetermined sequence, comprising:

a) contacting a polynucleotide comprising a 5′ portion and a 3′ portion, a template oligonucleotide having a 3′ blocker, a 5′ template portion, and a 3′ portion which is sufficiently complementary to the 3′ portion of the polynucleotide to hybridize thereto, and a polymerase under conditions in which the 3′ portion of the polynucleotide hybridizes to the 3′ portion of the template oligonucleotide and elongates to produce an elongated polynucleotide product; b) denaturing the duplex comprising the elongation product and a template oligonucleotide; c) repeating a) and b) in the presence of a second or subsequent template oligonucleotide to produce a second or subsequent elongation polynucleotide product.

Claims

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

a) combining in a mixture under conditions suitable for 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) one or more 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 the initial polynucleotide or to a 3′

portion of an extension polynucleotide formed by a polymerase extension reaction, to hybridize to the initial polynucleotide or to an extension polynucleotide wherein the initial polynucleotide and each of the one or more overlapping template oligonucleotides consist of not more than 100 contiguous nucleotides;

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

c) subjecting the mixture to denaturing conditions to separate extension polynucleotide and template oligonucleotide;

d) repeating b) and c).

2. A method according to claim 1 further comprising isolating the nucleic acid molecule from the mixture.

3. A method according to claim 1 wherein isolating the nucleic acid molecule from the mixture is by binding the nucleic acid to a column and eluting the nucleic acid from the column.

4. A method according to claim 1 wherein the nucleic acid molecule contains at least about 200 bases.

5. A method according to claim 4 wherein the nucleic acid molecule contains at least about 500 bases.

6. A method according to claim 1 wherein the initial polynucleotide comprises at least 20 contiguous nucleotides.

7. A method according to claim 1 wherein the one or more template oligonucleotides comprises at least 20 contiguous nucleotides.

8. A method 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.

9. A method according to claim 8, 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.

10. A method 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.

11. A method according to claim 1 wherein the repeating of b) and c) is for at least 10 cycles.

12. A method according to claim 11 wherein the repeating of b) and c) is for at least 20 cycles.

13. A method according to claim 1 wherein at least one of the one or more overlapping template oligonucleotides comprises a 3′

blocker.

14. A method according to claim 13 wherein the 3′

blocker is a 3′

, amino-substituted nucleotide or a dideoxy nucleotide.

15. A method according to claim 1 wherein the one or more template oligonucleotides comprise one oligonucleotide.

16. A method according to claim 15, wherein the one oligonucleotide has a sequence of at least about 8 contiguous nucleotides in the 3′

portion of the oligonucleotide, which is identical to a sequence of the same length in the 5′

portion of the oligonucleotide.

17. The method according to claim 16, wherein the identical 3′

and 5′

portion sequences of the second template oligonucleotide comprises at least about 15 nucleotides.

18. The method according to claim 16, wherein the identical 3′

and 5′

portion sequences of the second template oligonucleotide comprises at least about 25 nucleotides.

19. The method according to claim 1, wherein at least one of the an initial oligonucleotide, a template oligonucleotide, an extension polynucleotide, or a long nucleic acid molecule is attached to a solid phase support.

20. The method according to claim 19, wherein the initial polynucleotide is attached to the solid support.

21. The method according to claim 19, wherein the solid phase support is selected from a polymer matrix and a controlled-pore glass.

22. The method according to claim 19, wherein the at least one of the initial polynucleotide and one or more overlapping template oligonucleotides is attached at a 5′

end.

23. The method according to claim 19, wherein the at least one of the initial polynucleotide and one or more overlapping template oligonucleotides is attached at a 3′

end.

24. The method according to claim 19, wherein the at least one of the an initial oligonucleotide, a template oligonucleotide, an extension polynucleotide, or a long nucleic acid molecule attached to a solid phase support is attached through a linker moiety.

25. The method according to claim 24, wherein the linker moiety is selected from a phosphate linker and an amine linker.

26. The method according to claim 1, wherein a final long polynucleotide is amplified by the polymerase chain reaction.

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

a) contacting in a mixture, (i)a polynucleotide comprising a 5′

portion and a 3′

portion;

(ii) a template oligonucleotide having a 3′

blocker, a 5′

template portion, and a 3′

portion which is sufficiently complementary to the 3′

portion of the polynucleotide to hybridize thereto; and

(iii) a polymerase under conditions in which the 3′

portion of the polynucleotide hybridizes to the 3′

portion of the template oligonucleotide and the polynucleotide is elongated to produce an elongated polynucleotide product;

b) subjecting the mixture to denaturing conditions to separate the elongated polynucleotide product and the template oligonucleotide;

c) repeating a) and b) wherein the template oligonucleotide is a second or subsequent template oligonucleotide, wherein the elongated polynucleotide product is a first or subsequent elongated polynucleotide product, wherein the second or subsequent template oligonucleotide has a 3′

blocker, a 5′

template portion, and a 3′

portion which is sufficiently complementary to the 3′

portion of the first or subsequent elongated polynucleotide product to hybridize thereto, and wherein the first or subsequent elongated polynucleotide product is elongated to produce the nucleic acid molecule.

28. A method according to claim 27 wherein the nucleic acid molecule contains at least about 200 bases.

29. A method according to claim 28 wherein the nucleic acid molecule contains at least about 500 bases.

30. A method according to claim 27 wherein the polynucleotide comprises at least 20 contiguous nucleotides.

31. A method according to claim 30 wherein the polynucleotide comprises not more than 100 contiguous nucleotides.

32. A method according to claim 27 wherein the template oligonucleotide comprises at least 20 contiguous nucleotides.

33. A method according to claim 32 wherein the polynucleotide comprises not more than 100 contiguous nucleotides.

34. A method according to claim 27, wherein the 3′

portion of the 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 polynucleotide or an extension polynucleotide.

35. A method according to claim 34, wherein the 3′

portion of the 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 polynucleotide or an extension polynucleotide.

36. A method according to claim 27, wherein the 3′

portion of the 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 polynucleotide or an extension polynucleotide.

37. A method according to claim 27 wherein the repeating of a) and b) is for at least 10 cycles.

38. A method according to claim 37 wherein the repeating of a) and b) is for at least 20 cycles.

39. A method according to claim 27 wherein the 3′

blocker is an 3′

, amino-substituted nucleotide.

40. A method according to claim 27, wherein the 3′

blocker is a dideoxy nucleotide.

41. The method according to claim 27 wherein the second and subsequent template oligonucleotides are identical and the 3′

portions of the second template oligonucleotide comprises a sequence which is identical to a 5′

portion sequence of the same oligonucleotide such that elongation of the first or subsequent polynucleotide elongation product takes place along the 5′

template portion of the template oligonucleotides to produce second or subsequent polynucleotide elongation product having a 3′

portion which comprises a sequence complementary to the 3′

portions of the second template oligonucleotides.

42. The method according to claim 41, wherein the identical 3′

and 5′

portion sequences of the second template oligonucleotide comprises at least about 8 contiguous nucleotides.

43. The method according to claim 42, wherein the identical 3′

and 5′

portion sequences of the second template oligonucleotide comprises at least about 15 nucleotides.

44. The method according to claim 43, wherein the identical 3′

and 5′

portion sequences of the second template oligonucleotide comprises at least about 25 nucleotides.

45. The method according to claim 27, wherein at least one of (i) the polynucleotide comprising a 5′

portion and a 3′

portion and (ii) the template oligonucleotide having a 3′

blocker, a 5′

template portion, and a 3′

portion which is sufficiently complementary to the 3′

portion of the polynucleotide to hybridize thereto, is attached to a solid phase support.

46. The method according to claim 27, wherein the polynucleotide comprising a 5′

portion and a 3′

portion is attached to the solid support.

47. The method according to claim 27, wherein the solid phase support is selected from a polymer matrix and a controlled-pore glass.

48. The method according to claim 27, wherein the at least one of (i) the polynucleotide comprising a 5′

portion and a 3′

portion and (ii) the template oligonucleotide having a 3′

blocker, a 5′

template portion, and a 3′

portion which is sufficiently complementary to the 3′

portion of the polynucleotide to hybridize thereto, is attached at a 5′

end.

49. The method according to claim 27, wherein the at least(i) the polynucleotide comprising a 5′

portion and a 3′

portion and (ii) the template oligonucleotide having a 3′

blocker, a 5′

template portion, and a 3′

portion which is sufficiently complementary to the 3′

portion of the polynucleotide to hybridize thereto, is attached at a 3′

end.

50. The method according to claim 27, wherein the at least one of (i) the polynucleotide comprising a 5′

portion and a 3′

portion and (ii) the template oligonucleotide having a 3′

blocker, a 5′

template portion, and a 3′

portion which is sufficiently complementary to the 3′

portion of the polynucleotide to hybridize thereto to a solid phase support is attached through a linker moiety.

51. The method according to claim 50, wherein the linker moiety is selected from a phosphate linker and an amine linker.

52. The method according to claim 27, wherein a final long polynucleotide is amplified by the polymerase chain reaction.