Targeted Amplicon Genotyping by Sanger Sequencing.

This chapter presents a protocol for Sanger sequencing, a conventional yet rapid, cost-effective, and highly accurate method for targeted sequencing. It involves designing specific primers for forward and reverse template extension reactions, during which fluorophore labeled, chain terminating dideoxynucleotides are incorporated into newly replicated DNA molecules. The resulting end-labeled terminated products are separated by electrophoresis, enabling precise base identification within the amplicon. Chromatogram analysis further allows for the detection of nucleotide changes that may correspond to amino acid variations in targeted genes from different samples.