Sequencing

                         DNA sequencing is the determination of the order of nucleotides in a DNA molecule. The most popular method for doing this is called the Sanger sequencing method or chain-termination method. This method uses  synthetic nucleotides that lack the 3′ hydroxyl group and are unable to form the 3′–5′ phosphodiester bond necessary for chain elongation. These nucleotides are called dideoxynucleotides (ddNTPs). 

                          The sequencing reaction consists in a single-stranded template DNA to which a short complementary primer is annealed and extended by a DNA polymerase. The sequencing reaction contains a low concentration of ddNTPs, each labeled with a different fluorochrome, in addition to the normal deoxynucleotides. 

                           Once a ddNTP is incorporated in the elongating chain, it blocks further chain extension; as a result, a mixture of chains of lengths determined by the template sequence is accumulated. This mixture can then be resolved by CGE. CGE resolution allows separation of chains that differ in length by only one nucleotide.