Solid Phase PCR Sequencing of Biotinylated Products

The dideoxy sequencing method (1 ) has been universally employed and utilized for a wealth of sequencing projects such as small (2 ) and larger viral genomes (3 ), and requires purified M 13 or plasmid DNA templates. Numerous improvements have been made to obtain more DNA sequence from a single clone, including improved electrophorelic resolution and rapid computer assisted data entry. Inevitably, as these systems facilitate gel running and data entry, template preparation has become a limiting procedure. Attempts to improve this by the precipitation of M 13 phage with acetic acid and the recovery and subsequent disruption of the phage on glass fiber disks have been described (4 ). The DNA is eluted with a low salt buffer and requires neither phenol extraction nor ethanol precipitation, although bacterial culture and DNA purification are still required. Therefore, since the development of the polymerase chain reaction (PCR) (5 ), it has been used as a means of circumventing bacterial growth to prepare DNA templates. The main advantage is that template preparation becomes a simple biochemical process that can be readily automated if required. A major problem, though, is that the dNTPs and the two oligonucleotide primers from the PCR are present in great excess and must be removed either by electrophoresis or column chromatography (6 ).