Recombination of polynucleotide sequences using random or defined primers

• US 6,177,263 B1
• Filed: 07/14/1999
• Issued: 01/23/2001
• Est. Priority Date: 03/25/1997
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Giver, Lorraine J., Zhao, Huimin, Affholter, Joseph A., Shao, Zhixin, Arnold, Frances H.
• Primary Examiner(s): Schwartzman, Robert A.
• Assistant Examiner(s): LARSON, THOMAS GEORGE
• Application Number:
  US09/353,556
• Time in Patent Office: 559 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/69.1, 435/440, 435/455, 435/471, 435/489
• US Class Current: 506/11
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/54 bacteria being Bacillus
• CPC Class Codes: C12N 9/80 acting on amide bonds in linear amides (3.5.1)
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/155 Cyclic reactions
• CPC Class Codes: C12Q 2549/119 using nested primers

Abstract

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Claims

1. A method of recombining polynucleotides to produce recombinant polynucleotides, comprising:

(a) providing a population of different template polynucleotides having different sequences and a pair of first and second primers respectively disposed to hybridize to at least one strand of the template polynucleotides and its complement;

(b) conducting a multi-cyclic polynucleotide extension reaction on the template polynucleotides primed from the first primer and not the second primer under conditions of incomplete extension, whereby incomplete extension product primed from the first primer anneals to different members of the population of template polynucleotides having different sequences in different extension cycles and undergoes further extension thereby generating recombinant incomplete extension products and subsequently full-length recombinant extension products;

(c) contacting the full-length recombinant extension products with the second primer and extending the second primer to form double stranded full-length recombinant extension products.

2. The method of claim 1, wherein the template polynucleotides are single stranded.

3. The method of claim 2, wherein the template polynucleotides are of unknown sequence.

4. The method of claim 1, wherein the template polynucleotides comprise allelic variants.

5. The method of claim 1, further comprising amplifying the double stranded full-length recombinant extension product.

6. The method of claim 1, wherein the first primer is extended by an average of fewer than 300 nucleotides per extension cycle.

7. The method of claim 1, wherein the first primer is extended by an average of 20-50 nucleotides per extension cycle.

8. The method of claim 1, wherein the first and second primers are 6-100 nucleotides in length.

9. The method of claim 1, wherein at least one of the first and second primers comprises a random nucleotide at one or more nucleotide positions within the primer.

10. The method of claim 1, further comprising cloning the double stranded full-length recombinant extension products into multiple copies of a vector.

11. The method of claim 1, further comprising screening or selecting the double stranded full-length extension products or expression products thereof for an altered or enhanced property relative to the template polynucleotides or expression products thereof.

12. The method of claim 11, wherein the expression products are screened for an enzymatic activity.

13. The method of claim 1, wherein the template polynucleotides are RNA molecules, and the multi-cyclic polynucleotide extension reaction is conducted with a reverse transcriptase.

14. The method of claim 1, further comprising monitoring the formation of extension product at a plurality of times during the method.

15. A method of recombining polynucleotides to produce recombinant polynucleotides, comprising:

(a) providing a population of different template polynucleotides having different sequences and a pair of first and second primers respectively disposed to hybridize to at least one of the template polynucleotides and its complement;

(b) annealing the first primer to the at least one template polynucleotide;

(c) conducting a template dependent extension of the first primer under conditions of incomplete extension to form an incomplete extension product annealed to the at least one template;

(d) denaturing the incomplete extension product and the at least one template;

(e) annealing the incomplete extension product to at least a second template;

(f) conducting template dependent extension of the incomplete extension product to form a further incomplete or a full-length extension product, which extension product contains sequences extended from a plurality of the different templates having different sequences;

(g) denaturing the extension product of (f) and the template to which it is annealed;

(h) performing further cycles of (e-g) if needed to produce a full-length extension product;

(i) annealing the second primer to the full-length extension product of step (h), and extending the second primer to form a double stranded full-length extension product, the extension product being a recombined form of the template polynucleotides.

16. A method of recombining polynucleotides having different sequences, comprising:

performing in vitro recombination of first strands of a population of template polynucleotides having different sequences without recombining second strands complementary to the first strands;

synthesizing complementary strands to the recombined first strands to form recombined duplex polynucleotides.

17. The method of claim 16, wherein the different template polynucleotides are provided in single stranded form before performing the in vitro recombination.

18. The method of claim 16, wherein the different template polynucleotides are provided in double stranded form before performing the in vitro recombination.

19. The method of claim 16, wherein the different template polynucleotides comprise allelic variants.

20. The method of claim 16, further comprising amplifying the recombined duplex polynucleotides.

21. The method of claim 20, further comprising cloning the amplified recombined duplex polynucleotides into multiple copies of a vector.

22. The method of claim 16, further comprising screening or selecting the recombined duplex polynucleotides or expression products thereof for an altered or enhanced property relative to the template polynucleotides or expression products thereof.

23. The method of claim 22, wherein the expression products are screened or selected for an enzymatic activity.

24. The method of claim 16, wherein the template polynucleotides are of unknown sequence.

25. The method of claim 16, further comprising screening or selecting the recombined duplex polynucleotides or expression products thereof in a plate-based assay.

26. The method of claim 25, further comprising amplifying the recombined duplex polynucleotides.

27. The method of claim 26, further comprising cloning the amplified recombined duplex polynucleotides into multiple copies of a vector.

28. The method of claim 27, wherein the vector is a plasmid.

29. The method of claim 25, wherein the expression products are screened or selected for an enzymatic activity.

30. The method of claim 29, wherein the plate comprises a substrate for the enzymatic activity.

31. The method of claim 30, wherein the expression products are screened or selected for subtilisin activity.

32. The method of claim 30, wherein the expression products are screened or selected for deacylase activity.

33. The method of claim 28, wherein the expression products are screened or selected for an enzymatic activity.

34. The method of claim 33, wherein the plate comprises a substrate for the enzymatic activity.

35. The method of claim 34, wherein the expression products are screened or selected for subtilisin activity.

36. The method of claim 34, wherein the expression products are screened or selected for deacylase activity.

37. The method of claim 23, wherein the expression products are screened or selected for subtilisin activity.

38. The method of claim 23, wherein the expression products are screened or selected for deacylase activity.

39. The method of claim 16, wherein the in vitro recombination is performed with a mesophilic polymerase lacking strand displacement activity.

40. The method of claim 39, wherein said mesophilic polymerase lacking strand displacement activity is a bacteriophage DNA polymerase.

41. The method of claim 39, wherein the bacteriophage DNA polymerase is selected from the group consisting of T4 DNA polymerase and T7 Sequenase DNA polymerase.