Sequencing Using Custom Designed Oligonucleotides

Abstract

The dideoxy chain termination DNA sequencing procedure introduced in 1977 (1) has the advantage of being fast, simple to perform, and very accurate. Therefore, it became the method of choice to obtain several hundred bases of sequence information per reaction. The procedure is based on the enzymatic elongation and radioactive labeling of oligonucleotides that are complementary to the beginning of the single-stranded DNA template. Chain extension competes with the infrequent but specific termination by incorporation of a dideoxyribonucleotide. The products of four nucleotide-specific reactions can be separated on a polyacrylamide gel. An autoradiogram of such a gel finally provides the sequence information. Detailed descriptions of the various modifications of this method have been presented in preceding chapters of this volume. The synthetic oligonucleotide primer required for the synthesis of the labeled strands can easily be synthesized in automatic synthesizers or by hand, the latter of which is more laborious. Various so-called "universal sequencing" and "reverse sequencing" primers, complementary to the beginning of the polylinker region in M13 lac cloning phages and plasmids, are commercially available. Specifically designed oligonucleotides can also be prepared upon request by several molecular biological companies.