US5928905A
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US
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End-complementary polymerase reaction
Assignee: Glaxo Group Ltd
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Filing: 1996-07-03
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Granted: 1999-07-27
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Expires: 2016-07-03
Abstract
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.
Inventors
- 👤 Willem P. C. Stemmer
- 👤 Robert J. Lipshutz
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 05:07
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/63; C12N15/64; C12N15/66; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6853; C12Q1/686
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
131 words
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.
Claim 2
Independent Claim
131 words
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.
Claim 3
Dependent Claim
14 words
The method of claim 1, wherein the overlapping polynucleotides are provided in single-strand form.
Claim 4
Dependent Claim
26 words
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.
Claim 5
Dependent Claim
20 words
The method of claim 1, wherein the step of denaturation is performed at a temperature of at least 94 C.
Claim 6
Dependent Claim
17 words
The method of claim 1, wherein at least one of the overlapping polynucleotides comprises a variable-sequence kernel.
Claim 7
Dependent Claim
13 words
The method of claim 1, wherein the target polynucleotide is a circular vector.
Claim 8
Dependent Claim
14 words
The method of claim 1, wherein the target polynucleotide is at least 100 kb.
Claim 9
Dependent Claim
15 words
The method of claim 1, wherein the extended polynucleotides comprise concatemers of the target polynucleotide.
Claim 10
Dependent Claim
15 words
The method of claim 8, further comprising cleaving the concatemers to generate the target polynucleotide.
Claim 11
Dependent Claim
16 words
The method of claim 9, further comprising ligating the target polynucleotide to form circularized target polynucleotide.
Claim 12
Dependent Claim
24 words
The method of claim 1, further comprising transforming the extended polynucleotides into a cell whereby the extended polynucleotides recombine to form circularized target polynucleotide.
Claim 13
Dependent Claim
37 words
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.
Claim 14
Dependent Claim
11 words
The method of claim 1, wherein the overlapping polynucleotides are oligonucleotides.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2002-07-26 | 2003-01-25 | 2003-07-27 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2006-07-26 | 2007-01-25 | 2007-07-27 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2010-07-26 | 2011-01-25 | 2011-07-27 | $7700.00 | Window Closed / Lapsed |