Methods for recombinatorial nucleic acid synthesis

• US 6,994,963 B1
• Filed: 07/10/2000
• Issued: 02/07/2006
• Est. Priority Date: 07/10/2000
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Ambion Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Setterquist, Robert A., Murphy, George L., Ellington, Andrew D.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Wilder, Cynthia B.
• Application Number:
  US09/613,535
• Time in Patent Office: 2,038 Days
• Field of Search: 435/91.1, 435/91.2, 435/91.21, 435/6, 435/69.1, 435/91.51, 536/23.1, 536/23.2, 536/23.5, 536/24.3, 536/24.33
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

This invention pertains to a method for generating a pool of nucleic acid fragments useful for in vitro recombination and the creation of novel DNA sequences that encode desirable proteins or enzymes. The invention provides a defined mixture of nucleic acids and methods for use in the synthesis, mutagenesis, and recombination of nucleic acids. Nucleic acids may be synthesized by creating a nucleic acid extension ladder, annealing the extension ladder to template nucleic acids, and further extending the ladder of nucleic acids. The invention also relates to methods for performing repeated cycles of synthesis for the purpose of mutagenesis or recombination, methods for producing mutant peptides and proteins from the mutagenized or recombined nucleic acids, and methods for selecting a peptide, polypeptide or protein having altered biological activities.

Claims

1. A method for creating a plurality of nucleic acids comprising the steps of:

(a) annealing a plurality of a defined first primer nucleic acid to a plurality of at least one first single stranded template nucleic acid,

(b) performing a first extension by extending the first primer nucleic acid employing the first template nucleic acid in the presence of at least one dideoxynucleotide or a dideoxynucleotide comprising a nucleotide analog, wherein the dideoxynucleotide or dideoxynucleotide comprising a nucleotide analog is incorporated into the extended nucleic acid, to form a plurality of first extended nucleic acids having essentially identical 5′

ends and variable 3′

ends,

(c) denaturing the first extended nucleic acids from the first template nucleic acid,

(d) modifying or removing the dideoxynucleotide or dideoxynucleotide comprising a nucleotide analog from the first extended nucleic acids,

e) annealing the first extended nucleic acids to a plurality of at least a second single stranded template nucleic acid whose sequence is not identical to the first template nucleic acid, and

f) performing a second extension by extending the extended nucleic acid employing the second template nucleic acid to form a plurality of twice extended nucleic acids having essentially identical 5′

ends.

2. The method of claim 1, wherein the second extension is performed in the presence of at least one dideoxynucleotide or dideoxynucleotide analog, wherein the dideoxynucleotide or dideoxynucleotide comprising a nucleotide analog is incorporated into the extended nucleic acid, to form a plurality of twice extended nucleic acids having essentially identical 5′

ends and variable 3′

ends.

3. The method of claim 2, further comprising:

(a) modifying or removing the dideoxynucleotide or dideoxynucleotide comprising a nucleotide analog from the twice extended nucleic acids,

(b) denaturing the twice extended nucleic acid from the second template nucleic acid

(c) annealing the twice extended nucleic acid to a third template nucleic acid, and

(d) performing a third extension by extending the twice extended nucleic acid employing the third template nucleic acid to form a thrice extended nucleic acid.

4. The method of claim 3, further comprising adding at least one dideoxynucleotide or dideoxynucleotide comprising a nucleotide analog before or during the third extension.

5. The method of claim 3, further comprising at least one additional series of denaturing from a template, annealing to a further template, and performing of extension.

6. The method of claim 5, further defined as comprising between one and five hundred additional series of denaturing from a template, annealing to a further template, and performing of extension.

7. The method of claim 1, wherein said dideoxynucleotide or dideoxynucleotide comprising a nucleotide analog is removed by at least one exonuclease.

8. The method of claim 1, wherein said first single stranded template nucleic acid or said second single stranded template nucleic acid vary in size, sequence, resistance to cleavage or resistance to exonuclease degradation.

9. The method of claim 1, wherein said plurality of first extended nucleic acids comprises an extension ladder.

10. The method of claim 9, wherein said plurality of first extended nucleic acids vary in length, sequence, resistance to cleavage or resistance to exonuclease degradation.

11. The method of claim 10, wherein said plurality of first extended nucleic acids comprises nucleic acids of different sequence.

12. The method of claim 11, wherein said different sequence varies by one nucleotide.

13. The method of claim 10, wherein said plurality of first extended nucleic acids comprise different lengths.

14. The method of claim 13, wherein said different lengths comprise one nucleotide increments.

15. The method of claim 14, wherein said different lengths comprise more than one nucleotide increments.

16. The method of claim 1, wherein the extended nucleic acid comprises at least one partly double stranded nucleic acid or at least one fully double stranded nucleic acid.

17. The method of claim 1, wherein said defined first primer nucleic acid is resistant to cleavage or exonuclease digestion.

18. The method of claim 1, wherein said defined first primer nucleic acid is a plurality of primers.

19. The method of claim 18, wherein said plurality of primers vary in length, sequence, resistance to cleavage or resistance to exonuclease degradation.

20. The method of claim 1, wherein the first extended nucleic acid comprises the primer nucleic acid.

21. The method of claim 1, wherein said first or second extended nucleic acid is a recombinant, mutagenized or chimeric nucleic acid.

22. The method of claim 1, wherein said at least one first single stranded template nucleic acid or said at least one second single stranded template nucleic acid is a plurality of template nucleic acids.

23. The method of claim 1, further comprising the addition of at least one length-altering agent.

24. The method of claim 23, wherein the length-altering agent comprises a nucleotide, a nucleotide derivative, a nucleotide analog, a chemical treatment or a combination thereof.

25. The method of claim 24, wherein said length-altering agent comprises a nucleotide incorporated into said first or second extended nucleic acid.

26. The method of claim 25, wherein said nucleotide comprises at least one ribonucleotide.

27. The method of claim 26, wherein said length-altering agent further comprises treatment with an alkaline condition or a ribonuclease.

28. The method of claim 26, wherein said length-altering agent further comprises treatment with alkaline phosphatase and an exonuclease.

29. The method of claim 24, wherein said length-altering agent comprises a nucleotide derivative incorporated into said extended nucleic acid.

30. The method of claim 24, wherein the length-altering agent comprises a nucleotide analog incorporated into said extended nucleic acid.

31. The method of claim 30, wherein said nucleotide analog comprises at least one α

-phosphorothioate nucleotide.

32. The method of claim 31, wherein said length-altering agent further comprises alkylation of said extended nucleic acid.

33. The method of claim 24, wherein the length-altering agent comprises a chemical treatment of said extended nucleic acid.