DNA SEQUENCING TECHNOLOGY AND AUTOMATION

The precise manipulations of genetic material which are typical of contemporary experiments in molecular biology and the new biotechnology are based on the ability to determine DNA nucleotide sequences. This allows the advances made in genetics to be used for studying the complex processes of cell differentiation, and to intervene in cellular metabolism in a directed way, by creating transgenic organisms. This review concerns DNA sequencing technology, as found in wide use in 1991. The availability of reagents in kit form has made these techniques routine, almost "industrial" in nature. The present approach to DNA sequencing consists of recurrent breaking of genomic DNA into small fragments, which are then amplified and sequenced, and the original sequence is then reconstructed from the sequences of the overlapping fragments. DNA sequencing consists of a number of steps: amplification and purification of the desired DNA fragment, preparation of DNA in a form suitable for the selected method of sequencing, the sequencing experiments themselves, fractionation of the reaction products, interpretation of the results of each sequencing experiment (e.g., reading autoradiographs), and reconstruction of the original sequence from the sequences of the overlapping short fragments. These steps are considered individually, most attention being paid to sequencing strategies applicable to given biological problems. The review ends with a discussion of automation of sequencing experiments and a consideration of problems associated with the development of DNA sequencing in the USSR.