A Laboratory Guide to Genomic Sequencing - The Direct Sequencing of Native Uncloned DNA

A Safety Considerations Genomic sequencing involves a number of hazardous steps, such as high current, high voltage, radioactive and highly toxic chemicals. It is, therefore, absolutelyessen- tial that the instructions of equipment manufacturers be followed and that particular attention is paid to the local and federal safety regulations. INTRODUCTION 9 B Introduction During the cloning of genomic DNA many of its characteristics are perma- nently lost. It was therefore necessary to develop a new technique that would give us a closer look at a gene in its normal environment. The powerful technique of genomic sequencing, first described by Church and Gilbert (1984) now makes it possible to have a precise view of a given DNA sequence in a chromosome. This method combines the chemical DNA-sequencing procedure of Maxam and Gilbert (1980) with the detection of DNA sequences by electroblotting and indirect end-labeling by hybridization. Besides studies on the methylation state of single bases in a given gene (Nick et al., 1986; Saluz and Jost, 1986; Saluz et al., 1986), genomic sequencing can also be used to study specific DNA-protein interactions in vivo (Church et al., 1985; Giniger et al., 1985; Becker et al., 1986; Ephrussi et al., 1985; Martin et al., 1986; Nick et al., 1986; Zinn and Maniatis, 1986).