DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Effect of pyrophosphorolysis and metal ions

J Biol Chem. 1990 May 15;265(14):8322-8.


Pyrophosphorolysis by bacteriophage T7 DNA polymerase leads to the degradation of specific dideoxynucleotide-terminated fragments on DNA sequencing gels. This reaction can be prevented by pyrophosphatase. It is also inhibited by a high concentration of dNTPs; only the dNTP complementary to the next base in the template is an effective inhibitor, suggesting the formation of a stable polymerase-primer-template-nucleotide complex despite the absence of a 3' hydroxyl group on the primer. The use of pyrophosphatase, a genetically modified T7 DNA polymerase that lacks exonuclease activity, and Mn2+ rather than Mg2+ to eliminate discrimination between dideoxynucleotides and deoxynucleotides (Tabor, S., and Richardson, C. C. (1989) Proc. Nat. Acad. Sci. U. S. A. 86, 4076-4080) generates bands of uniform intensity on a DNA sequencing gel. Uniform band intensities simplify the analysis of a DNA sequence, particularly with automated procedures. For example, when genomic DNA is sequenced directly, heterozygotic sequences are readily detected because their bands have half the intensity of homozygotic sequences. A procedure for automated DNA sequencing is described that exploits the uniformity. A single reaction with a single labeled primer is carried out using four different ratios of dideoxynucleotides to deoxynucleotides; after gel electrophoresis in a single lane, the sequence is determined by the relative intensity of each band.

Publication types

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

MeSH terms

  • Animals
  • Autoanalysis
  • Base Sequence
  • DNA / metabolism*
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyadenine Nucleotides / metabolism
  • Deoxycytosine Nucleotides / metabolism
  • Deoxyguanine Nucleotides / metabolism
  • Deoxyribonucleotides / metabolism
  • Dideoxynucleotides
  • Drosophila / genetics
  • Genetic Carrier Screening
  • Magnesium / pharmacology
  • Manganese / pharmacology*
  • Molecular Sequence Data
  • Mutation
  • Phosphates / metabolism*
  • Polymerase Chain Reaction
  • Pyrophosphatases / pharmacology
  • T-Phages / enzymology*
  • Thymine Nucleotides / metabolism


  • Deoxyadenine Nucleotides
  • Deoxycytosine Nucleotides
  • Deoxyguanine Nucleotides
  • Deoxyribonucleotides
  • Dideoxynucleotides
  • Phosphates
  • Thymine Nucleotides
  • 2',3'-dideoxyadenosine triphosphate
  • Manganese
  • 2',3'-dideoxycytidine 5'-triphosphate
  • 2',3'-dideoxyguanosine 5'-triphosphate
  • DNA
  • bacteriophage T7 induced DNA polymerase
  • DNA-Directed DNA Polymerase
  • Pyrophosphatases
  • Magnesium
  • 2',3'-dideoxythymidine triphosphate