Efficient PCR Production of Single-Stranded DNA Sequencing Templates

The acceptance of polymerase chain reaction (PCR) as a routine method in molecular biology has created a need for simple and robust approaches to DNA sequencing of the amplification products. In addition to its ease, direct sequencing of PCR products has the advantage that nucleotide misincorporation during amplification does not pose a problem, because only a small percentage of the DNA molecules may contain such random mutations. However, a large body of published protocols attests to the difficulties encountered in this endeavor. Double-stranded sequencing of the linear PCR products is notoriously unreliable because of rapid reannealing of the denatured DNA.