DNA Sequencing

For generation of different sized DNA fragment, two methods are generally used. One is Sanger’s method in which dideoxyribonucleotide triphosphates are used to terminate DNA synthesis at different sites. The other method is known as Maxam-Gilbert method in which DNA threads are chemically cut into pieces of different sizes.

DNA sequencing (as shown in the figure) is usually done by copying a strand of the cloned DNA in four different reaction mixtures, using a modified form of DNA polymerase. In each reaction mixture the copies are made so that the newly synthesized DNA chains are terminated randomly at positions corresponding to one of the four basis. The lengths of the fragments from each reaction are then determined by gel electrophoresis, allowing the sequence of base in the cloned DNA fragment to read of from one end to the other.

The volume of DNA sequence is now large that powerful computers must be used to store and analyze it. DNA sequence is now completely automated, robotic devices mix the reagents and then load, run and read the order of the nucleotide basis from the gel. This is facilitated by using chain terminating nucleotide that are each labelled with a different colored fluorescent dye; in this case, all four synthesis reactions can be performed in the same tube, and the products can be separated in a single lane of a gel. A detector position near the bottom of the gel reads and record the color of fluorescent label on each band as it passes through a LASER beam. A computer then reads and stores this nucleotide sequence.