Sequencing of DNA

After cloning and amplification of DNA it can be analyzed for its sequence determination. The manual enzymatic method (sanger) can be used where ddNTS are used which can terminate DNA strand synthesis at specific nucleotides as the strand is synthesized on purified template nucleic acid. Here radiolabelled primers are used. One can separate fragments according to size using PAGE. Another method is of Maxam and Gilbert, employs chemical method to cleave the DNA molecules where they contain the specific nucleotides.

Automated DNA sequencing is another approach. Here four different fluorescent labels – one representing each nucleotide are used. Each emits a specific signal upon excitation by laser beam of particular wavelength, which can be recorded by computer. This is the most widely used method.

(Source: Harper's Illustrated Biochemistry)