Health Information

Determination of nucleotide sequence in a DNA molecule is the basic and fundamental requirement in biotechnology. DNA sequencing is important to understand the functions of genes, and basis of inherited disorders. Further, DNA cloning and gene manipulation invariably require knowledge of accurate nucleotide sequence.

MAXAM and GILBERT technique

The first DNA sequencing technique, using chemical reagents, was developed by maxam and gilbert (1977). This method is briefly described below

A strand of source DNA is labeled at one end with 32p. The two strands of DNA are then separated. The labeled DNA is distributed into four samples in separate tubes. Each sample is subjected to treatment with a chemical that specifically destroys one (G, C) or two bases (A+G, T+C) in the DNA. Thus, the DNA strands are partially digested in four samples at sites G, A+G, T+C and C. This result in the formation of a series of labeled fragment depends on the site at which the base is destroyed from the labeled end. Thus for instance, if there are C residues at position 44,7, and 10 away from the labeled end, then treatment of DNA that specifically destroys C will give labeled pieces of length 3, 6 and 9 bases. The labeled DNA fragments obtained in the four tubes are subjected to electrophoresis side by side and they are detected by autoradiograph. The sequence of the bases in the DNA can be constructed from the bands on the electrophoresis.

Dideoxynucleotide method

Currently, the preferred technique for determining nucleotide sequence in DNA is the one developed by Sanger (1980). This is an enzymatic procedure commonly referred to as the Dideoxynucleotide method or chain termination method.

A Dideoxynucleotide is a laboratory-made chemical molecule that lacks a hydroxyl group at both the 2’ and 3’ carbons of the sugar. This is in contrast to the natural deoxyribonucleotide that possesses at 3’ hydroxyl group on the sugar.