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Sanger sequencing

1st sequencing – Sanger sequencing

  • Sanger sequencing by capillary electrophoresis is the gold-standard DNA sequencing technique.
  • Based on the selective incorporation of chain-terminating ddNTP by DNA polymerase during in vitro DNA replication.
  • requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleosidetriphosphates (dNTPs), and modified di-deoxynucleosidetriphosphates (ddNTPs)
  • resulted DNA fragments are heat denatured and separated by size using gel electrophoresis
  • DNA polymerases copy single-stranded DNA templates by adding nucleotides to a growing chain. Chain elongation occurs at the 3' end of a primer, an oligonucleotide that anneals to the template. The deoxynucleotides added to the extension product are selected by base-pair matching to the template.
  • Thermal cycling -> purification -> capillary electrophoresis

http://media.invitrogen.com.edgesuite.net/ab/applications-technologies/pharma-biotherapeutics/DNA_sequencing.swf

https://en.wikipedia.org/wiki/Sanger_sequencing