Rapid and reliable dideoxy sequencing of double-stranded DNA

Gene. 1985;40(2-3):317-23. doi: 10.1016/0378-1119(85)90055-1.

Abstract

We report a simple and reliable protocol for nucleotide sequencing using the Sanger dideoxy technique on linearized double-stranded DNA molecules with specific oligonucleotide primers. The method is demonstrated for restriction fragments cloned into the plasmid vectors pSP64 and pSP65 using two vector-specific primers, the M13 reverse primer and a new SP6 primer, flanking the multiple cloning site. Template DNA may be prepared by a rapid alkaline lysis procedure. Mild linearization conditions with the appropriate restriction endonuclease avoid the appearance of artifact bands.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cloning, Molecular
  • DNA Restriction Enzymes
  • DNA, Recombinant*
  • Genetic Vectors
  • Phosphorus Radioisotopes
  • Plasmids
  • Templates, Genetic

Substances

  • DNA, Recombinant
  • Phosphorus Radioisotopes
  • DNA Restriction Enzymes