ABSTRACT
DNA is a complex molecule made of smaller units (nucleotides). DNA sequencing plays an important role in genetics. Total nucleic acid sequencing was discovered by Robert Holley and his colleagues in 1965. In 1972, Walter Fiers completed the first gene sequencing of bacteriophage MS2 using the 2-D fractionation technique. A few years later in 1975, Sanger and his assistant, Alan Coulson, introduced the “plus/minus system”; and Allan Maxam and Walter Gilbert followed that up with the chemical cleavage technique in 1976–1977. In the Sanger method (also called chain-termination or dideoxy method), the DNA strand (chain) is terminated when one of four dideoxynucleotides (ddNTPs), which lack the 3’ OH group, becomes incorporated. The resulting sequences are run separately on polyacrylamide gel, producing bands of different sizes. At the end, the gel is exposed to either UV light or X-rays. It reads smaller fragments less than one kilo base (kb). Currently advance techniques like polymerase chain reaction, recombinant DNA technologies, and revolutionary genomics provide the means of producing the optimum concentration of pure DNA species demanded by high-quality sequencing. The existing Sanger sequencing technique is the backbone of genome sequencing, including microbial sequencing. The aim of this chapter to is to explain the Sanger sequencing method in detail with some examples.
