[Amplification of the phage lambda DNA sequence by polymerase chain reaction using thermostable DNA polymerase]

Mol Biol (Mosk). Nov-Dec 1991;25(6):1602-10.
[Article in Russian]

Abstract

The efficiency of phage DNA amplification by the method of polymerase chain reaction (PCR) with Tth DNA-polymerase was studied for optimization of PCR conditions. The effect on amplification efficiency of medium ionic strength and pH, the presence of univalent cations, detergents, gelatin, ATP, pyrophosphate, SH-reagents and ratio of concentrations of Mg and dNTPs, primers and template was studied. It has been found that a pH optimum for PCR with Tth DNA-polymerase varies from 8.5 to 9.0. An ionic strength optimum for PCR is about 0.08. The influence of univalent cations on the activity of Tth DNA-polymerase can be expressed as NH4+ greater than Na+ greater than K+. 0.01% Tween-20 significantly increases the efficiency of PCR and 0.01% gelatin inhibits it. Addition of ATP, pyrophosphate, SH-reagents to the reaction mixture did not increase the yield of PCR product. It has been also shown that for the given PCR-system an optimum Mg/dNTPs molar ratio is within the range of 1.5-2.0. An optimum concentration of each of the pair of primers for this PCR-system is about 0.3 microM. The possibility of PCR-amplification of 500-8500 b.p. DNA fragments has been demonstrated.

Publication types

  • English Abstract

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Cations
  • DNA, Viral / genetics*
  • DNA-Directed DNA Polymerase / metabolism*
  • Detergents
  • Diphosphates / metabolism
  • Gene Amplification*
  • Genes, Viral
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Osmolar Concentration
  • Polymerase Chain Reaction
  • Sulfhydryl Compounds / metabolism

Substances

  • Cations
  • DNA, Viral
  • Detergents
  • Diphosphates
  • Sulfhydryl Compounds
  • Adenosine Triphosphate
  • DNA-Directed DNA Polymerase