Methods of synthesizing polynucleotides using thermostable enzymes

• US 20050064490A1
• Filed: 09/23/2004
• Published: 03/24/2005
• Est. Priority Date: 09/23/2003
• Status: Abandoned Application

• Current Assignee: Curators of the University of Missouri (University of Missouri)
• Original Assignee: Curators of the University of Missouri (University of Missouri)
• Inventors: Oleksiak, Marjorie F., Crawford, Douglas L.
• Application Number:
  US10/947,832
• Publication Number:
  US 20050064490A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions

Abstract

Disclosed are methods of synthesizing a polynucleotide complementary to a target polynucleotide include steps of subjecting a non-thermophilic cell comprising a thermostable polymerase to a temperature effective to disrupt the cell to form a reaction mixture, wherein the reaction mixture comprises the target polynucleotide and one or more primers that hybridize to a sequence of the target polynucleotide or to a sequence flanking the polynucleotide and incubating the reaction mixture under conditions whereby the polynucleotide is synthesized. Also disclosed are cell libraries and kits in accordance with the invention.

Claims

1. A method of synthesizing a polynucleotide complementary to a target polynucleotide comprising:

a) subjecting a non-thermophilic cell comprising a thermostable polymerase to a temperature effective to disrupt the cell to form a reaction mixture, wherein the reaction mixture comprises the target polynucleotide and one or more primers that hybridize to a sequence of the target polynucleotide or to a sequence flanking the target polynucleotide; and

b) incubating the reaction mixture under conditions whereby a polynucleotide complementary to at least a portion of the target polynucleotide is synthesized.

2. The method of claim 1, wherein the cell comprises a polynucleotide encoding the thermostable polymerase operably connected to a promoter functional in the cell.

3. The method of claim 2, wherein the polynucleotide encoding the thermostable polymerase is integrated into the genome of the cell.

4. The method of claim 2, wherein the cell comprises a cloning vector comprising a sequence encoding the thermostable polymerase.

5. The method of claim 4, wherein the cloning vector further comprises the target polynucleotide.

6. The method of claim 1, wherein the cell comprises a cloning vector comprising the target polynucleotide.

7. The method of claim 1, wherein the cell comprises a first cloning vector comprising a sequence encoding the thermostable polymerase, and wherein the cell further comprises a second cloning vector comprising the target polynucleotide.

8. The method of claim 1, wherein the reaction mixture further comprises one or more primers that hybridize to the complementary polynucleotide synthesized in step b).

9. The method of claim 1, wherein the reaction mixture comprises one or more deoxyribonucleotides, ribonucleotides or dideoxynucleotides.

10. The method of claim 1, wherein the reaction mixture comprises a reaction buffer.

11. The method of claim 1, wherein the reaction mixture comprises a cloning vector comprising the target polynucleotide.

12. The method of claim 11, wherein a second non-thermophilic cell comprises the cloning vector.

13. The method of claim 11, wherein a virus comprises the cloning vector.

14. The method of claim 11, wherein the cloning vector is linearized.

15. The method of claim 11, wherein the cloning vector is episomal.

16. The method of claim 1, wherein the thermostable polymerase is a reverse transcriptase, an RNA polymerase or a DNA polymerase.

17. The method of claim 1, wherein the thermostable polymerase is selected from polymerases natively expressed in Thermococcus litoralis, Bacillus stearothermophilus, Pyrococcus furiosus, Pyrococcus woesei, Thermus aquaticus, Thermus filiformis, Thermus flavus, Thermus thermophilus or Thermotoga maritema or recombinant variants thereof.

18. The method of claim 1, wherein the conditions of step b) include thermal cycling.

19. The method of claim 1, wherein the reaction mixture further comprises a DNA helicase.

20. The method of claim 1, wherein the cell is a prokaryotic cell.

21. The method of claim 20, wherein the prokaryotic cell is E. coli.

22. The method of claim 1, wherein the cell is a eukaryotic cell.

23. The method of claim 22, wherein the cell is a mammalian cell.

24. The method of claim 1, wherein the cell is a yeast cell.

26. A kit for synthesizing a polynucleotide according to the method of claim 1 comprising:

a population of non-thermophilic cells, at least one cell in the population comprising a polynucleotide encoding a thermal stable polymerase.

27. The kit of claim 26, further comprising a polynucleotide comprising a cloning vector.

28. The kit of claim 26, wherein the cells are competent cells.

29. The kit of claim 27, further comprising one or more primers that hybridize to the cloning vector.

30. The kit of claim 27, wherein the cloning vector comprises a multi-cloning sequence.

31. The kit of claim 26, further comprising at least one reaction buffer.

32. The kit of claim 26, further comprising one or more deoxyribonucleotides, ribonucleotides or dideoxynucleotides.

33. The kit of claim 32, wherein at least one of the deoxyribonucleotides, ribonucleotides or dideoxynucleotides comprises a detectable label.

34. The kit of claim 26, further comprising at least one restriction endonuclease.

35. The kit of claim 26, further comprising a ligase.

36. The kit of claim 26, further comprising a DNA helicase.

37. The kit of claim 26, further comprising instructions for use of the kit.

25. A library comprising a population of non-thermophilic cells comprising a plurality of target polynucleotides, at least one cell in the population comprising a polynucleotide encoding a thermostable polymerase.