DNA sequencing means methods for determining the order of the nucleotides bases adenine, guanine, cytosine and thymine in a molecule of DNA. This method is based on nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides.
The first DNA sequence was obtained by academic researchers, using laboratories methods based on 2- dimensional chromatography in the early 1970s.
By the development of dye based sequencing method with automated analysis, DNA sequencing has become easier and faster.
In 1976-1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases.
Base-specific cleavage of DNA by some specific chemicals
Four different chemicals, one for each base
A set of DNA fragments of different sizes
DNA fragments contain up to 500 nucleotides
Purified DNA can be read directly
Homopolymeric DNA runs are sequenced as efficiently as heterogeneous DNA sequences
Can be used to analyze DNA protein interactions such as footprinting
Can be used to analyze nucleic acid structure and epigenetic modifications to DNA
Maxam–Gilbert sequencing requires radioactive labeling at one 5′ end of the DNA fragment to be sequenced (typically by a kinase reaction using gamma-32P ATP) and purification of the DNA. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using hydrazine. The addition of salt (sodium chloride) to the hydrazine reaction ceases the reaction of thymine for the C-only reaction. The modified DNAs may then be cleaved by hot piperidine; (CH2)5NH at the position of the modified base. The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule.
The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation.
To read the sequence, we begin with the smaller fragments at the bottom of the gel. “Calling” each base involves interpreting the band pattern relative to the four chemical reactions. For example, if a band in the DNA sequence appears in both the G-reaction and the G+A-reaction lanes, then that the nucleotide is a G. If a band in the DNA sequence appears only in the G+A-reaction lane, then it is an A. The same decision process works for the C-reaction and the C+T-reaction lanes. Sequences are confirmed by running replicate reactions on the same gel and comparing the autoradiographic patterns between replicates.
Although this method is based on very simple principles but it has s lot of troubles. Using this method we could only confirm about 200–300 bases of DNA every few days.
A few probles are;
The radioactive labeling process
The cleavage reactions
Setting of gel
The electrophoresis process
The X-ray film developer