Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1988-05-18
1989-10-10
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
435 6, 435 68, 435 91, 4351721, 4353171, 435320, 935 10, 935 16, 536 27, C12N 1500, C12N 700, C12P 2100, C12P 1934
Patent
active
048731927
ABSTRACT:
The present invention discloses several DNA mutagenesis processes using a DNA template containing several uracil residues in place of thymine, which can be applied without selection techniques to produce altered DNA sequences with approximately 10-fold greater efficiency than current methods of site-specific mutagenesis.
This template has relatively normal coding potential in the in vitro reactions typical of standard site-directed mutagenesis protocols but is not biologically active upon transfection into a wild type (i.e., ung.sup.+) E. coli host cell. Expression of a desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus favored. The procedure has been applied to mutations introduced via both obligonucleotides and error-prone polymerization. The inclusion of two additional simple treatment steps before transfection results in a site-specific mutation frequency approaching 100%.
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Jain Mishrilal
Roberts, Jr. John S.
Seidman S.
The United States of America as represented by the Department of
Wiseman Thomas G.
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