15.12: Genome Sequencing I - Traditional Methods
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. Coincidentally, in the same year, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. Both of these methods rely on the separation of a mixture of DNA fragments of variable size using a polyacrylamide gel and deciphering the DNA sequence from the resulting gel bands.
Sanger Sequencing Vs.The Maxam-Gilbert Method
Sanger sequencing can be used to sequence roughly 700-800 bp of DNA in a single run, which is comparatively higher than the Maxam-Gilbert Method, which typically resolves up to 400bp. Additionally, the requirement of relatively large amounts of template DNA, coupled with the use of radioisotopes and hazardous chemicals, makes the Maxam-Gilbert method less favorable for routine use. However, because of the direct use of purified DNA, the Maxam-Gilbert method is more accurate than Sanger sequencing.
Present Day Applications of Traditional Sequencing Methods
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. Although the Maxam-Gilbert method is less widely used, it is still preferred for use in some techniques, such as DNA fingerprinting and DNA structural studies.