Long-range PCR with a DNA polymerase fusion

Methods Mol Biol. 2011;687:17-23. doi: 10.1007/978-1-60761-944-4_2.

Abstract

Proofreading DNA polymerase fusions offer several advantages for long-range PCR, including faster run times and higher fidelity compared with Taq-based enzymes. However, their use so far has been limited to amplification of small to mid-range targets. In this article, we present a modified protocol for using a DNA polymerase fusion to amplify genomic targets exceeding 20 kb in length. This procedure overcomes several limitations of Taq blends, which up until recently, were the only option for long-range PCR. With a proofreading DNA polymerase fusion, high-molecular-weight amplicon can be generated and analyzed in a single day, and a significant proportion is expected to be error-free.

MeSH terms

  • Base Sequence
  • DNA Primers
  • DNA-Directed DNA Polymerase / metabolism*
  • Polymerase Chain Reaction / methods*

Substances

  • DNA Primers
  • DNA-Directed DNA Polymerase