Sanger sequencing is a method of determining the nucleotide order of DNA that involves incorporating a modified nucleotide (dideoxynucleotides) that inhibits the function of DNA polymerase during in vitro DNA replication.
There are three main steps:
1. Generate DNA fragments of varying lengths, each terminated with a labelled dideoxynucleotide. Each of the four DNA bases is tagged with a different label.
2. Separate the DNA sequences by length using capillary gel electrophoresis. The shorter fragments move faster than the longer fragments.
3. Excite the label dideoxynucleotide at the end of each sequence with a laser generating a chromatogram showing the fluorescent peak of each nucleotide. The chromatogram has the nucleotides in the correct order because of the electrophoresis.