Applied Biosystems/3730XL
Up until very recently, the primary sequencing platform at the HGSC was the AB 3730XL. The platform is based on classical Sanger dideoxy-nucleotide protocols in which plasmid subclone libraries are amplified by growth in E. coli followed by purification using an alkaline lysis plasmid prep to generate double stranded templates. Templates may also be generated by PCR amplification. Cycle sequencing reactions containing template, primer, polymerase, unlabeled nucleotides and fluorescently-labeled BigDye TerminatorTM dideoxy-nucleotides results in the generation of single-stranded products of varying length, each of which ends in one of the four dye-labeled bases complementary to its corresponding position in the template. The reactions are loaded on the sequencing instrument and 96 reactions per run are separated by size using capillary electrophoresis. Emerging from the capillaries, the fluorescent tags are excited by a laser and recorded using a CCD camera. Successive rounds of technology development over the years have resulted in read lengths approaching 1000 bases using as little as 1/128th volume of reaction mix.
Second generation sequencing platforms promise vastly greater sequence volume at much reduced cost and amplification of templates without the use of bacteria.
