End-complementary polymerase reaction

• US 5,928,905 A
• Filed: 07/03/1996
• Issued: 07/27/1999
• Est. Priority Date: 04/18/1995
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Glaxo Group Limited (GSK plc), Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Lipshutz, Robert J., Stemmer, Willem P. C.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/675,502
• Time in Patent Office: 1,119 Days
• Field of Search: 435/91.2, 435/91.1, 435/91.53, 536/22.1, 536/24.3
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

The present invention is directed to a process for amplifying and detecting any target nucleic acid sequence contained in a nucleic acid or mixture thereof and for assembling large polynucleotides from component polynucleotides, each involving generating concatemers formed by PCR amplification of overlapping fragments.

Claims

1. A method for amplifying a target polynucleotide, comprising

(1) providing a series of overlapping polynucleotides of alternating polarity spanning the target polynucleotide, wherein each given polynucleotide in the series has two flanking polynucleotides, and the 3'"'"' end of the given polynucleotide is complementary to the 3'"'"' end of one flanking polynucleotide and the 5'"'"' end of the given polynucleotide is complementary to the 5'"'"' end of the other flanking polynucleotide;

(2) incubating the polynucleotides under conditions whereby complementary ends anneal;

(3) extending the 3'"'"' ends of annealed polynucleotides with a polymerase, under conditions whereby each polynucleotide serves as a template for another;

(4) denaturing the extended polynucleotides; and

(5) repeating steps (2)-(4) 1-100 times, whereby the extended polynucleotides in step (4) constitute the polynucleotides in step (2) in the next cycle.

2. The method of claim 1, wherein the overlapping polynucleotides are provided in single-strand form.

3. The method of claim 1, wherein 3+ end of the annealed polynucleotides are extended by a polymerase which is Taq polymerase, TthI polymerase or Vent polymerase.

4. The method of claim 1, wherein the step of denaturation is performed at a temperature of at least 94 C.

5. The method of claim 1, wherein at least one of the overlapping polynucleotides comprises a variable-sequence kernel.

6. The method of claim 1, wherein the target polynucleotide is a circular vector.

7. The method of claim 1, wherein the target polynucleotide is at least 100 kb.

8. The method of claim 1, wherein the extended polynucleotides comprise concatemers of the target polynucleotide.

9. The method of claim 8, further comprising cleaving the concatemers to generate the target polynucleotide.

10. The method of claim 9, further comprising ligating the target polynucleotide to form circularized target polynucleotide.

11. The method of claim 1, further comprising transforming the extended polynucleotides into a cell whereby the extended polynucleotides recombine to form circularized target polynucleotide.

12. The method of claim 1, wherein the overlapping polynucleotides are provided as component strands of double stranded polynucleotides and the method further comprises denaturing the double stranded polynucleotides before the incubating step whereby the complementary ends anneal.

13. The method of claim 1, wherein the overlapping polynucleotides are oligonucleotides.