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US 5,928,905 AGranted
End-complementary polymerase reaction
AssigneesGlaxo Group Limited, Affymetrix, Inc.
InventorsWillem P. C. Stemmer, Robert J. Lipshutz
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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.
Claims13
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.
3 more elements
Dependencies are inferred from the claim text.