METHODS OF SYNTHESIZING POLYNUCLEOTIDES USING THERMOSTABLE ENZYMES

• US 20060252083A1
• Filed: 06/26/2006
• Published: 11/09/2006
• 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:
  US11/426,422
• Publication Number:
  US 20060252083A1
• 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 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.

2. A kit for synthesizing a polynucleotide complementary to at least a portion of a target polynucleotide, the kit comprising:

(a) a population of non-thermophilic cells, at least one cell in the population comprising a thermal stable polymerase; and

(b) instructions for;

(i) subjecting the non-thermophilic cells to a temperature effective to disrupt the cells to form a reaction mixture comprising the thermal stable polymerase and the target polynucleotide; and

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

3. The kit of claim 2, further comprising a polynucleotide comprising a cloning vector.

4. The kit of claim 2, wherein the cells are competent cells.

5. The kit of claim 3, further comprising one or more primers that hybridize to the cloning vector.

6. The kit of claim 3, wherein the cloning vector comprises a multi-cloning sequence.

7. The kit of claim 2, further comprising at least one reaction buffer.

8. The kit of claim 2, further comprising one or more deoxyribonucleotides, ribonucleotides or dideoxynucleotides.

9. The kit of claim 8, wherein at least one of the deoxyribonucleotides, ribonucleotides or dideoxynucleotides comprises a detectable label.

10. The kit of claim 2, further comprising at least one restriction endonuclease.

11. The kit of claim 2, further comprising a ligase.

12. The kit of claim 2, further comprising a DNA helicase.

13. The kit of claim 2, wherein the instructions further comprise incubating the reaction mixture with one or more primers that hybridize to a sequence of the target polynucleotide or to a sequence flanking the target polynucleotide.

14. The kit of claim 2, wherein the instructions further comprise introducing the target polynucleotide into at least one cell in the population of non-thermophilic cells prior to subjecting the non-thermophilic cells to a temperature effective to disrupt the cells.