DNA sequencing with chain-terminating inhibitors

This paper describes a further method using DNA polymerase, which makes use of inhibitors that terminate the newly synthesized chains at specific residues

F. Sanger; S. Nicklen; A. R. Coulson

2006

Scholarcy highlights

  • A new method for determining nucleotide sequences in DNA is described
  • This paper describes a further method using DNA polymerase, which makes use of inhibitors that terminate the newly synthesized chains at specific residues
  • In this experiment only dideoxynucleoside triphosphates were used as inhibitors because the results with araC were much less satisfactory when a restriction enzyme was used for the subsequent splitting
  • After annealing of the template and primer a single ribonucleotide is incorporated by incubation with DNA polymerase in the presence of manganese and the appropriate ribonucleoside triphosphate
  • The method described here has a number of advantages over the plus and minus methods
  • For the longer sequences in which the separate bands in a run are not resolved, experience has shown that it is frequently possible to estimate the number of nucleotides from the strength and width of the band
  • The most serious difficulties are due to "pile-ups" of bands, which are usually caused by the DNA forming base-paired loops under the conditions of the acrylamide gel electrophoresis

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