[91] Fluorescent and radioactive solid-phase dideoxy sequencing of polymerase chain reaction products in microtiter plates

This chapter focuses on the fluorescent and radioactive solid-phase dideoxy sequencing of polymerase chain reaction (PCR) products in microtiter plates. The reactions presented are easy to perform and are reproducible, using various different sources of DNA template. Both short and long PCR products can be readily sequenced and oligo walking along the solid-phase-bound template is demonstrated. A further advantage of solid-phase sequencing is the high quality of the sequence data produced. The chapter discusses the development of a robust technique for directly sequencing terminally biotinylated PCR products by selective immobilization to streptavidin-coated magnetic beads. This technique can be applied with different labeling approaches either [α-35S] deoxyadenosine triphosphate (dATP), fluorescently labeled dideoxynucleoside triphosphates, or fluorescently labeled sequencing primers. It has been used to generate DNA sequences amounting in excess of 200 kb in total. The method is applicable to short and long templates, and the semi automation of the technique is described in this chapter.