DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

28. Development and Evaluation of a PCR-Based Sequencing Routine for Use on the ABI 3700 Capillary Machine

Lynne Goodwin, Owatha Tatum, Olga Chertkov, Judith Cohn, and P. Scott White

Bioscience Division and DOE Joint Genome Institute, Los Alamos National Laboratory, Los Alamos, NM 87545

swhite@telomere.lanl.gov

The rapid throughput of the new generation of capillary electrophoresis instruments for automated DNA sequencing presents unique problems for high throughput applications. Increased demands for sequencing substrate of high quality place strains on template preparation routines and equipment, and alternative strategies that scale well and cost less are needed. We have implemented a PCR-based strategy for creating sequencing template that requires only a few, easily automated steps, and costs a fraction of commercial robotic plasmid prep protocols. The template production process begins with overnight culture of subclones, followed by pin-stamp inoculation of PCR plates (96 or 384-well), and finishes with an enzymatic treatment of the PCR products to provide suitable sequencing template. Forward and reverse sequencing reactions are then performed directly on these templates using vector primers, followed by a single isopropanol precipi-tation, and resuspension in 1/10 TE. The 1/10 TE resuspension buffer allows samples to remain on the capillary instrument at ambient temperature for extended periods of time, which is necessary to obtain essentially hands-off automated sequencing of 6 to 8 96-well plates in a 24 hr period.

Concerns about the quality of sequence derived from PCR templates generally focus on homopolymer and simple repeat stretches that are difficult to accurately reproduce using Taq DNA polymerase. In addition, the number of failed reactions and the read length of PCR template-generated sequence is thought to be less than sequence generated from highly purified plasmid DNA. If the costs are favorable and the data of sufficient quality, then such a template production strategy is attractive; otherwise, the higher up front cost of plasmid template preparation is money well spent, as long as throughput is not limited. We have examined the quality of data obtained from an ABI 3700 using this process, and directly compared subclone sequence generated from plasmid templates on ABI 377 instruments. The results will be presented, as will a discussion of the implementation of this process.