15.13: Sanger Sequencing
DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a mixture of DNA fragments of variable size using capillary gel electrophoresis and deciphering the DNA sequence from the resulting electropherogram.
Challenges of Sanger Sequencing
Sanger sequencing can only be used to sequence roughly 300-1000 bp of DNA in a single run. The quality of the Sanger sequence at the primer binding site which makes the first 15 to 40 nucleotides in a sequence is poor.
Present Day Applications of Sanger Sequencing
Due to its simplicity and reliability, the conventional Sanger sequencing technique was quickly adapted into a semi-automated method to make it more accurate, reliable, and fast. Today, it is frequently used for small-scale targeted sequencing.