In vitro mutagenesis of Xanthomonas campestris alpha-amylase gene by partially replacing deoxythymidine triphosphate with 5-bromo-2'-deoxyuridine-5'-triphosphate using a PCR technique

Biotechnol Lett. 2004 Jan;26(2):171-5. doi: 10.1023/b:bile.0000012901.89522.20.

Abstract

Three mutants of the wild type alpha-amylase gene from Xanthomonas campestris pv. campestris 8004 were obtained using a PCR technique in which deoxythymidine triphosphate (dTTP) was partially replaced by 5-bromo-2'-deoxyuridine-5'-triphosphate (BrdUTP), at an optimal dTTP:BrdUTP ratio of 1000:1. Of thre three mutants that were obtained and which were sequenced, one mutant with 40 times higher activity than the wild type alpha-amylase gene product was obtained by using primary PCR products as a template for a second PCR reaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular
  • Deoxyuracil Nucleotides / chemistry*
  • Mutagenesis*
  • Polymerase Chain Reaction
  • Thymine Nucleotides / chemistry*
  • Xanthomonas campestris / genetics*
  • alpha-Amylases / genetics*

Substances

  • Deoxyuracil Nucleotides
  • Thymine Nucleotides
  • bromodeoxyuridine triphosphate
  • alpha-Amylases
  • thymidine 5'-triphosphate