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Explain the method of DNA sequencing using automated DNA sequencing technique.

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Automatic DNA Sequencers: Automatic sequencing machines were developed during 1990’s. It is an improvement of Sanger’s method. In this new method, a different fluorescent dye is tagged to the ddNTP’s. Using this technique, a DNA sequence containing thousands of nucleotides can be determined in a few hours. Each dideoxynucleotide is linked with a fluorescent dye that imparts different colors to all the fragments terminating in that nucleotide. All four labelled ddNTP’s are added to a single capillary tube. It is a refinement of get electrophoresis which separates fastly. DNA fragments of different colors are separated by their size in a single electrophoretic gel.

A current is applied to the gel. The negatively charged DNA strands migrate through the pores of gel towards the positive end. The small sized DNA fragments migrate faster and vice-versa. All fragments of a given length migrate in a single peak. The DNA fragments are illuminated with a laser beam. Then the fluorescent dyes are excited and emit light of specific wavelengths which is recorded by a special ‘recorder’. The DNA sequences are read by determining the sequence of the colours emitted from specific peaks as they pass the detector. This information is fed directly to a computer which determines the sequence. A tracing electrogram of emitted light of the four dyes is generated by the computer. Colour of each dye represents the different nucleotides. Computer converts the data of emitted light in the nucleotide sequences.

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