US 7,399,590 B2 | ||
Recombinase polymerase amplification | ||
Olaf Piepenburg, St. Albans (United Kingdom); Colin H. Williams, St. Albans (United Kingdom); Niall A. Armes, London (United Kingdom); and Derek L. Stemple, St. Albans (United Kingdom) | ||
Assigned to ASM Scientific, Inc., Cambridge, Mass. (US) | ||
Filed on Sep. 01, 2004, as Appl. No. 10/931,916. | ||
Application 10/931916 is a continuation in part of application No. 10/371641, filed on Feb. 21, 2003, granted, now 7,270,981. | ||
Claims priority of provisional application 60/553999, filed on Mar. 16, 2004. | ||
Claims priority of provisional application 60/358563, filed on Feb. 21, 2002. | ||
Prior Publication US 2005/0112631 A1, May 26, 2005 | ||
Int. Cl. C12Q 1/68 (2006.01); C12P 19/34 (2006.01) |
U.S. Cl. 435—6 [435/91.1; 435/91.2] | 190 Claims |
1. A recombinase polymerase amplification process of DNA amplification of a double stranded target nucleic acid molecule,
said target nucleic acid molecule comprising a first and second strand of DNA, comprising the steps of
(a) contacting a recombinase agent selected from the group consisting of uvsX and recA, with a first and second nucleic acid
primer to form a first and second nucleoprotein primer, wherein said nucleic acid primer comprises a single stranded region
at its 3′ end;
(b) contacting the first and the second nucleoprotein primer to said double stranded target nucleic acid molecule thereby
forming a first double-stranded structure at a first portion of said first strand and forming a second double stranded structure
at a second portion of said second strand such that the 3′ ends of said first nucleic acid primer and said second nucleic
acid primer are oriented toward one another on the same double-stranded template nucleic acid molecule;
(c) extending the 3′ end of said first and second nucleic acid primer with one or more polymerases and dNTPs to generate a
first and second double-stranded nucleic acid and a first and second displaced strands of nucleic acid; and
(d) continuing the reaction through repetition of (b) and (c) until a desired degree of amplification is reached, wherein
said process at steps (b) and (c) is performed in the presence of a crowding agent such that the crowding agent stimulates
amplification.
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