The sequence of a deoxyribonucleic acid DNA molecule can be elucidated using chemical or enzymatic methods.
Chemical Method of DNA Sequencing:
In 1997, Maxam and Gilbert of Harward University discovered this method. In this method, a DNA fragment to be sequenced is radiolabeled at one end of molecule (Fig. 13.15). After denaturation of DNA into single strand, bases are modified by chemical treatment and are divided into four reaction samples.
In the first reaction tube guanine base is modified. Whereas in second reaction tube, both adenine and guanine are modified. Similarly, in the third reaction tube only cytosine is modified.
In the fourth reaction tube both thymine and cytosine bases are modified. All the modified bases in few reaction mixtures are then subjected to second chemical treatment which brings breakage at the backbone of the modified bases.
This breakage results in the production of various lengths of DNA molecule. All the DNA fragments of variable lengths are separated by gel electrophoresis and the interpretation is that DNA bands on the gel differ from one another by single base.
Enzymatic Method of DNA Sequencing:
This method was discovered by Fred Sanger in 1977. This method is also called enzymatic method or chain termination method. The enzymatic method of sequencing is based on the ability of a DNA polymerase to extend primer, after hybridized to the template that it is to be extended until a chain-termination nucleotide is incorporated.
In the sequencing process of terminating a single-stranded, DNA fragment to be sequenced is allowed to bind with a short radiolabeled, complementary fragment called a primer.
Following this, primer bound fragments is then divided into four separate reactions, each of which contains all four deoxyribonucleotide triphosphates (dNTP’s) supplemented with a limiting amount of a different dideoxyribonucleotide triphosphate (ddNTP), because ddNTPs lack the 3′-OH group necessary for chain elongation, the growing oligonucleotide is terminated selectively at G, A, T or G, depending on which dideoxy analog present in the reaction.
The cleave adjustment in relative concentrations of each of the dNTPs and ddNTPs can set termination of chain. The resulting fragments, each with a common origin but ending in a different nucleotide, are separated according to size by denaturing poly acrylamide gel electrophoresis and finally subjected to autoradiography (Fig. 13.14).
