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Abstract

This chapter discusses deoxyribonucleic acid (DNA) sequencing using α-thiodeoxynucleotides. The chapter describes a set of protocols used currently in laboratories for sequence analysis, involving both a modification of previously described subcloning techniques to create ordered sets of templates, as well as sequencing reactions using the α -thiodeoxynucleoside triphosphate substitution technique for sequence analysis. The chapter mentions the sources of the materials that are needed, such as enzymes and nucleotides. The chapter discusses the production of ordered deletion libraries, size screening of deletion clones, preparation of templates for sequencing, and so on. In the production of ordered deletion libraries, the replicative form of recombinant M13 is subjected to limited digestion with DNase I so that only a relatively small fraction of all DNA molecules is linearized. The principle of the thio sequencing method is outlined in the chapter. After annealing the universal sequencing primer to the template DNA, the second strand is synthesized by the addition of mixtures of thiodeoxy-and deoxynucleoside triphosphates.