Sequencing Double-stranded DNA

It is also possible to undertake direct DNA sequencing from doublestranded molecules such as plasmid cloning vectors and PCR amplicons. The double-stranded DNA must be denatured prior to annealing with
primer. In the case of plasmids, an alkaline denaturation step is sufficient. However, for PCR amplicons this is more problematic. Unlike plasmids, amplicons are short and reanneal rapidly. Denaturants such as formamide and dimethyl sulfoxide have been used to prevent the reannealing of PCR strands following their separation. Another strategy is to bias the amplification towards one strand by using a primer ratio of 100:1, which also overcomes this problem to a certain extent.
It is possible physically to separate and retain one PCR product strand by incorporating a molecule such as biotin into one of the primers. Following PCR, the strand that contains the biotinylated primer may be removed by affinity chromatography with streptavidin-coated magnetic beads, leaving the complementary PCR strand. This magnetic affinity purification provides single-stranded DNA derived from the PCR amplicon and, although somewhat time consuming, it does provide high-quality single-stranded DNA for sequencing.