What is Sanger sequencing and what is it used for?

Also known as the chain termination method, Sanger sequencing is a form DNA sequencing, which is the determination of the nucleotide order of DNA. Sanger sequencing utilizes dideoxynucleotides, which differ from normal nucleotides (deoxynucleotides) by lacking a hydroxyl group on the 3’ carbon. The lack of the hydroxyl group prevents another nucleotide from being added, thus terminating the sequence.

In Sanger sequencing, PCR is conducted using a reaction of primers, DNA polymerase, nucleotides, and dye-tagged dideoxynucleotides, along with the DNA sample to be sequenced. This result in fragments of the DNA sample terminated at each possible position in its sequence. Then, using capillary gel electrophoresis, fragments are sorted by length and identified using the dyes’ fluorescence, thereby revealing the nucleotide sequence of the DNA sample.