Fully-automated, nonradioactive solid-phase sequencing of genomic DNA obtained from PCR.

Abstract

Nonradioactive sequencing in combination with solid-phase template purification is a powerful method for sequence analysis, especially of PCR-generated fragments. To perform DNA sequencing under optimal conditions, it is necessary to obtain a well-purified and single-stranded (ss) DNA template. The disadvantage of ssDNA sequencing is that additional steps are required to generate ssDNA templates before any sequencing reactions can be carried out and is thus more time-consuming. Here we describe an automated solid-phase system for direct sequencing of ssPCR products that incorporates magnetic beads coated with streptavidin as solid support and a computer-controlled device (PolySeq) with heating, magnetic and mixing functions, all of which are integrated into a robotic workstation (Biomek 1000). This solid-phase method is extremely useful for rapid template purification and strand separation of DNA obtained from PCR as well as for high-quality dideoxyribonucleotide chain termination sequencing with fluorescently labeled primers. The system allows the complete automation of the sequencing procedure starting with PCR amplicons. DNA sequencing results obtained with this system were reproducible and gave an excellent length of readable sequences with low background and an analysis capacity of 30,000-40,000 bp per week.