Silver Sequencing: Nonradioactive Cycle Sequencing of dsDNA

The conventional methods of sequencing, such as Maxam and Gilbert (1 ) (involving chemical cleavage of labeled DNA fragments) and Sanger et al. (2 ), of dideoxy sequencing (using enzymatic extension of oligonucleotide primers) remain the most reliable techniques, but when compared to more modern methods, they are most labor-intensive. For rapid sequence analysis of limited quantities of template, cycle sequencing (3 ) has become increasingly popular. The technology, described in detail by Blakesley (4 ), is basically as other dideoxy sequencing techniques, but using a thermostable polymerase, Taq polymerase (5 ), which is resistant to multiple heat denaturation steps. This allows repriming from the same template, resulting in a linear increase in the quantity of synthesized product.