MANIFOLD SEQUENCING - EFFICIENT PROCESSING OF LARGE SETS OF SEQUENCING REACTIONS

Automated instruments for DNA sequencing greatly simplify data collect ion in the Sanger sequencing procedure. By contrast, the so-called fro nt-end problems of preparing sequencing templates, performing sequenci ng reactions, and loading these on the instruments remain major obstac les to extensive sequencing projects. We describe here the use of a ma nifold support to prepare and perform sequencing reactions on large se ts of templates in parallel, as well as to load the reaction products on a sequencing instrument. In this manner, all reaction steps are per formed without pipetting the samples. The strategy is applied to seque ncing PCR-amplified clones of the human mitochondrial D-loop and for d etection of heterozygous positions in the human major histocompatibili ty complex class II 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.