8] The use of DNase I, buffer gradient gel, and 35s label for DNA sequencing

Abstract

This chapter discusses deoxyribonucleic acid (DNA) sequencing through the use of DNase I, buffer gradient gel, and 35 s label. Buffer gradient gels and 35 s label are simple means of increasing the rate of sequence analysis, because they add little time to that required for determining the sequences of a given number of clones, increasing the amount of useful data per gel. The method of generating sequential deletion subclones with DNaseI in the dideoxy chain termination procedure has made it possible to read sequence data progressively along the length of DNA. Starting from an M13 RF recombinant DNA with a 2-kb insert, it normally takes a few days to obtain two sets of sequential deletion subclones, which are sufficient to cover the whole length of the insert DNA in both orientations. The DNase I procedure can be used to generate mutations of target genes by insertion of synthetic restriction enzyme linkers into the breakage site caused by DNase I cleavage of a plasmid containing a target gene. The chapter discusses the generation of sequential deletion subclones by DNase I. A buffer gradient can be used for reducing the vertical band spacing on a polyacrylamide sequencing gel. The chapter mentions several related concepts, including transfection into M13, identification of the original clone, construction of sequential deletion subclones, and so on.