PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates

Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216-20. doi: 10.1073/pnas.91.6.2216.


A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template.

MeSH terms

  • Bacteriophage lambda / genetics*
  • Base Composition
  • Base Sequence
  • DNA Primers
  • DNA, Viral / analysis*
  • DNA, Viral / chemistry
  • Exonucleases / metabolism
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Mutation
  • Particle Size
  • Polymerase Chain Reaction / methods*
  • Templates, Genetic


  • DNA Primers
  • DNA, Viral
  • Exonucleases