Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated PCR

Methods Enzymol. 2002;350:258-77. doi: 10.1016/s0076-6879(02)50968-x.

Abstract

Each of the adaptamer-directed genome manipulation methods is predicated on the fact that recombination between two DNAs is enhanced by increasing the length of homology. Many of the current PCR-based genome manipulation techniques rely on very short homologies to promote recombination. In these cases homology length is dictated by the technical limits of oligonucleotide synthesis. Adaptamers circumvent this problem since long homology regions are produced in a first round of PCR, and then fused to the selectable marker in a second round of PCR via complementary sequence tags on the adaptamers. Furthermore, many of the techniques described here rely on preexisting and commercially available adaptamer sets that can be obtained inexpensively rather than designing new primers for every experiment. Although a cost is incurred when performing multiple PCR amplifications, the increase in recombination efficiency is dramatic. Finally, the adaptamer-mediated PCR fusion methodology is versatile and can be applied to varied genome manipulations.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA Primers
  • Genetic Markers
  • Genome, Fungal*
  • Mutagenesis*
  • Polymerase Chain Reaction / methods
  • Recombinant Proteins / genetics
  • Saccharomyces cerevisiae / genetics*

Substances

  • DNA Primers
  • Genetic Markers
  • Recombinant Proteins