Title:
POLYMERASE-BASED PROTOCOLS FOR THE INTRODUCTION OF DELETIONS AND INSERTIONS
Document Type and Number:
WIPO Patent Application WO2004072252
Kind Code:
A3
Abstract:
The present invention relates to improved methods of introducing site-directed mutations into circular DNA without the need for subcloning. The invention comprises single-stage, polymerase-based, mutagenesis procedures which eliminate the need for the second-stage linear cyclic amplification reaction of the prior art. In accordance with the invention, production of complimentary mutagenized DNA strands is accomplished in separate, single-primer, linear amplification reactions carried out for each primer of a mutagenic primer pair. The mutagenized DNA strands produced in the separate primer reactions are combined and annealed and the resulting double-stranded mutagenized DNA intermediate may then be used directly to transform a host cell for further production of the desired mutant DNA. Major applications of this method include directed evolution and other areas that benefit from the development of diversity.
Inventors:
SALERNO JOHN C (US)
Application Number:
PCT/US2004/003616
Publication Date:
December 16, 2004
Filing Date:
February 06, 2004
Export Citation:
Assignee:
RENSSELAER POLYTECH INST (US)
SALERNO JOHN C (US)
SALERNO JOHN C (US)
International Classes:
C07H21/04; C12N15/00; C12N15/10; C12N15/64; C12N15/66; C12N15/70; C12P19/34; C12Q1/68; C12N; (IPC1-7): C12N15/00; C07H21/04; C12N15/70
Domestic Patent References:
| WO1997020950A1 | 1997-06-12 |
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