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Automated instruments for DNA sequencing greatly simplify data collection in the Sanger sequencing procedure. By contrast, the so-called front-end problems of preparing sequencing templates, performing sequencing reactions, and loading these on the instruments remain major obstacles to extensive sequencing projects. We describe here the use of a manifold support to prepare and perform sequencing reactions on large sets of templates in parallel, as well as to load the reaction products on a sequencing strument. In this manner, all reaction steps are performed without pipetting the samples. The strategy is applied to sequencing PCR-amplifled clones of the human mitochondrial D-loop and for detection of heterozygous positions in the human major histocompatibility complex class H gene HLA-DQB, amplified from genomic DNA samples. This technique will promote sequencing in a clinical context and could form the basis of more efficient genomic sequencing strategies.Large-scale genomic sequencing projects now under way are paralleled by an increasing interest to determine the nucleotide sequences of specific genes in patient samples. These efforts have prompted the development of a number of automated instruments to record nucleotide sequence data (1, 2). Other time-consuming aspects of collecting nucleotide sequence information, besides recording the information, include preparing the sequencing templates and generating and loading the sequencing reaction products on gels. Solidphase sequencing of PCR products is an efficient method of preparing sequencing templates that results in high-quality sequence data (3, 4). We demonstrate in this paper a solidphase-based method to process, in parallel, large sets of reactions up to and including the point where the sequencing reaction products are loaded on an automated sequencing instrument. In this strategy, sequencing templates are isolated and handled by using a solid support with 96 prongs projecting into individual reaction wells. Of crucial importance for our approach was the development of a very efficient means of immobilizing amplified sequencing templates on the limited surface available on the manifold support. This effect was achieved by creating a porous surface layer on the polystyrene device (5). In this manner, sufficient avidin can be coupled to individual prongs of the support in order to bind and sequentially transfer sets of biotin-labeled templates for fluorescence sequencing reactions through the series of reaction steps preceding gel analysis (Fig. 1). MATERIALS AND METHODSSolid Supports. Comb-shaped devices, similar to those used when casting sequencing gels to create slots for loading samples, were cut out of sheets of 100-,m biaxial polystyrene (Nordbergs Tekniska AB, Sundbyberg, Sweden) (Fig. 1A). As an alternative, a commercially available polystyrene device was used. This support has a set of 96 ball-and-stick extensions, projecting into individual wells of a microtiter plate located underneath (Falcon).Avidin-coated supports were constructe...

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