A multisite-directed mutagenesis using T7 DNA polymerase: application for reconstructing a mammalian gene
- PMID: 3224824
- DOI: 10.1016/0378-1119(88)90380-0
A multisite-directed mutagenesis using T7 DNA polymerase: application for reconstructing a mammalian gene
Abstract
A method to introduce multiple mutations and to reconstruct genes, using a single oligodeoxyribonucleotide and DNA polymerase with high processivity, such as modified T7 DNA polymerase [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 84 (1987a) 4767-4771], is described. A eukaryotic cDNA, coding for porcine growth hormone (pGH), was reconstructed in this study to delete 75 bp and to introduce a G----A transition. The deletion removes 75 bp and brings an ATG just upstream from the codon for the first amino acid in the mature protein. Moreover, the G----A substitution creates a new PvuII restriction site to facilitate further manipulation of the gene. Maximum mutation frequency with this multisite-directed mutagenesis is reached within 15 min with an efficiency approaching 50%, when using the modified T7 DNA polymerase. No multisite-directed mutants were obtained when T4 DNA polymerase or Klenow (large) fragment of DNA polymerase I were used. The described method is also applicable to simple single site-directed mutations as well as to more complex gene reconstruction strategies.
Similar articles
-
A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA.Gene. 1992 Nov 2;121(1):17-24. doi: 10.1016/0378-1119(92)90157-k. Gene. 1992. PMID: 1427092
-
Genetic analysis of the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin.Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9774-8. doi: 10.1073/pnas.89.20.9774. Proc Natl Acad Sci U S A. 1992. PMID: 1409697 Free PMC article.
-
DNA substrate structural requirements for the exonuclease and polymerase activities of procaryotic and phage DNA polymerases.Biochemistry. 1989 Mar 7;28(5):1975-83. doi: 10.1021/bi00431a004. Biochemistry. 1989. PMID: 2541768
-
DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Effect of pyrophosphorolysis and metal ions.J Biol Chem. 1990 May 15;265(14):8322-8. J Biol Chem. 1990. PMID: 2159476
-
A method for unidirectional deletion mutagenesis with application to nucleotide sequencing and preparation of gene fusions.Gene. 1986;49(1):119-28. doi: 10.1016/0378-1119(86)90391-4. Gene. 1986. PMID: 3552883
Cited by
-
The complex bet promoters of Escherichia coli: regulation by oxygen (ArcA), choline (BetI), and osmotic stress.J Bacteriol. 1996 Mar;178(6):1655-62. doi: 10.1128/jb.178.6.1655-1662.1996. J Bacteriol. 1996. PMID: 8626294 Free PMC article.
-
The E2 signal sequence of rubella virus remains part of the capsid protein and confers membrane association in vitro.J Virol. 1990 Nov;64(11):5500-9. doi: 10.1128/JVI.64.11.5500-5509.1990. J Virol. 1990. PMID: 2214022 Free PMC article.
-
Noninducible Tet repressor mutations map from the operator binding motif to the C terminus.J Bacteriol. 1993 Feb;175(4):1206-10. doi: 10.1128/jb.175.4.1206-1210.1993. J Bacteriol. 1993. PMID: 8432715 Free PMC article.
-
Production of infectious recombinant Moloney murine leukemia virus particles in BHK cells using Semliki Forest virus-derived RNA expression vectors.Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11658-63. doi: 10.1073/pnas.93.21.11658. Proc Natl Acad Sci U S A. 1996. PMID: 8876192 Free PMC article.
-
Incorporation of homologous and heterologous proteins into the envelope of Moloney murine leukemia virus.J Virol. 1994 Aug;68(8):4879-89. doi: 10.1128/JVI.68.8.4879-4889.1994. J Virol. 1994. PMID: 8035486 Free PMC article.
Publication types
MeSH terms
Substances
Associated data
- Actions
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
