A multisite-directed mutagenesis using T7 DNA polymerase: application for reconstructing a mammalian gene.
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.
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Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
el-Gewely
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D000818:Animals; D001483:Base Sequence; D002872:Chromosome Deletion; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005796:Genes; D013006:Growth Hormone / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010902:Pituitary Gland / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D013552:Swine; D013604:T-Phages / Q000201:enzymology*
D015345:Oligonucleotide Probes; D013006:Growth Hormone; D004259:DNA-Directed DNA Polymerase