Solid-phase sequencing of biotinylated PCR products with streptavidin-coated magnetic beads

A novel approach to generating high-quality single-stranded DNA involves solid-phase sequencing. A biotin group is incorporated at the 5'-end of one of the amplification primers and as a result becomes incorporated into PCR product during the amplification reaction. The DNA can then be immobilized onto streptavidin-coated paramagnetic beads, simultaneously removing buffers, dNTPs, and unincorporated PCR primers. By alkali denaturation, the immobilized double-stranded DNA is converted into a single-stranded template, which can then be purified away from its complementary strand, avoiding any competition in the subsequent sequencing reaction. Although the protocol detailed in this chapter is designed to purify and sequence PCR products, the method is broadly applicable to the solid-phase sequencing of many other templates, e.g., plasmid DNA.