15.12: 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.

For certain experiments or diagnoses, it may be necessary to obtain the nucleotide sequence of the entire genome of an organism to gain a deeper understanding of the genes or alleles present, their function, and associated diseases. This can be achieved using DNA sequencing.

Two well-known traditional DNA sequencing techniques are the Sanger sequencing method and the Maxam-Gilbert method.

In Sanger sequencing - also known as chain termination or dideoxynucleotide sequencing - the pure template DNA to be sequenced is PCR amplified in the presence of appropriate primers, dNTPs and modified bases, called dideoxynucleotides or ddNTPs.

The dideoxynucleotides lack the hydroxyl group of deoxynucleotides, which limits their ability to form phosphodiester bonds with adjacent nucleotides.

Each dideoxynucleotide is also labeled with a different fluorescent label to make its detection easier. 

The first step is to denature the template DNA into a single-stranded DNA.

Then, as the primers bind to the region of interest, the DNA polymerase starts adding new nucleotides to the DNA strand and occasionally incorporates a dideoxynucleotide instead of a deoxynucleotide - which terminates the DNA amplification.

The result is DNA fragments of varying lengths, each terminating with a labeled dideoxynucleotide.

These DNA fragments are then run on a capillary gel to separate them on the basis of size, and the emission spectra from each of these fragments are analyzed through automated software to decipher the genetic sequence of the desired DNA.