15.12 : Sanger 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.