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