Site-directed mutagenesis of DNA

Details Site-directed mutagenesis of DNA Site-directed mutagenesis of DNA

1993-08-11

1994-10-11

Parr, Margaret

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435 6, 435 912, 435 914, 935 16, 935 17, 935 79, 935 84, C12P 1934

Patent

active

053546709

ABSTRACT:
This invention pertains to a method of site-specific mutagenesis of DNA. The method can be used to mutagenize DNA, especially circular DNA (for example, virtually any plasmid), requiring only that the DNA carry a nonessential, unique recognition site for a restriction enzyme (restriction site). According to the method, a parental DNA to be mutated, containing a nonessential, unique restriction site, is used to generate progeny DNA containing the desired mutation but lacking the restriction site. The non-mutant parental-type DNA and the mutant progeny DNA are treated with any enzyme that cleaves at the restriction site to cleave the parental-type DNA, leaving the mutant DNA uncleaved. Mutant progeny DNA is selected by transforming cells with the enzyme-treated DNA under conditions such that the cells are transformed with uncleaved DNA at a higher efficiency than cleaved DNA with the result that the majority of transformants carry the mutant DNA. In preferred embodiments, the DNA is circular. The restriction enzyme treatment linearizes parental DNA which retains the nonessential, unique restriction site, leaving mutant DNA which lacks the site circular. Circular DNA transforms host cells more efficiently than linear DNA.

REFERENCES:
Marotti et al Gene Anal Techs (1989) 6:67-70.
Nells et al. Gene (1985) 34:315-323.
Rochlitz et al. DNA (1988) 7:515-519.
Lewis et al Nucleic Acids Res (1996) 18:3439-3443.
Pharmacia LKB Biotechnology Products Catalog, 1989.
Higuchi, R. Using PCR to engineer DNA. 1989. In: PCR Technology: Principles and Applications for DNA Amplifications. (H. Erlich, Editor) Stockton Press, New York, N.Y. pp. 61-70.
Kriegler, M. Electroporation. 1990. Gene Transfer and Expression: A Laboratory Manual. Stockton Press, New York, N.Y., pp. 101-102.
Altered Sites.RTM. In Vitro Mutagenesis System, (1993) Promega Catalogue p. 138.
International Search Report for International Application No. PCT/US92/11076, completed Feb. 3, 1993, mailed Feb. 23, 1993.
Vandeyar et al. (1988) Gene 65:129-133.
Masumune et al. (1971) J. Biol. Chem. 246:2692-2701.
Nossal (1974) J.Biol. Chem. 249:5668-5676.
Corden et al. (1980) Science 209:1406-1414.
Charles et al. (1982) J. Biol. Chem. 257:7930-7932.
Villafranca et al. (1983) Science 222:782-788.
Gillam et al. (1984) J. Virol. 52:892-896.
Kunkel (1985) Proc. Natl. Acad. Sci. (USA) 82:488-492.
Kunkel et al. (1987) Meth. Enzymol. 154:367-382.
Smith (1985) Ann. Rev. Genet. 19:423-462.
Mandecki (1986) Proc. Natl. Acad. Sci. (USA) 83:7177-7181.
Carter (1987) Meth. Enzymol. 154:382-403.
Zell et al. (1987) EMBO J. 6:1809-1815.
Saiki et al. (1988) Science 239:487-491.
Lewis et al. (1990) Nucleic Acids Res. 18:3439-3443.
Deng et al. (1992) Anal. Biochem. 200:81-88.
Ray et al. (1992) Biotechniques (in press).
Gillam et al. (1985) J. Virol. 53:708-709.

Affiliated with

Also associated with

Rating

Site-directed mutagenesis of DNA does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Site-directed mutagenesis of DNA, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Site-directed mutagenesis of DNA will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-1658040