Bi-directional Sanger Sequence analysis

Sanger sequencing is a first generation sequencing method. The process consists of a selective incorporation chain-terminating dideoxynucleotides ((ddNTPs) by DNA polymerase during in vitro DNA replication. Classical Sanger sequencing requires a single-stranded DNA template, a DNA polymerase, a DNA primer, normal deoxynucleosidetriphosphates (dNTPs), and fluorescently labeled modified nucleotides (ddNTPs) that terminate DNA strand elongation. During primer elongation, the random insertion of a ddNTP instead of a dNTP terminates synthesis of the chain because DNA polymerase cannot react with the missing hydroxyl. The products are separated on a single lane capillary gel, where the resulting bands are read by a imaging system. In bi-directional Sanger Sequence analysis, analyses are performed simultaneously with two opposing primers.