HPLC purification of PCR products for direct sequencing

The usefulness of sequence data derived from cloned PCR products may be limited by cloning artefacts and the cloning of minor sequence variants (e.g. ex vivo HIV). Direct sequencing of PCR-amplified fragments avoids these problems, and can also clarify where artefacts may have been introduced by Taq polymerase. However, PCR products larger than 200 bp cannot usually be sequenced directly without further purification. Such PCR products may be purified by gel electrophoresis. Alternatively, products can be purified by ethanol precipitation or by using microconcentrator columns (e.g. Centricon super(TM)) which, although fast and very popular, may not yield consistent results; moreover, neither of these methods can separate individual PCR products. It is possible to purify PCR products by HPLC. We have adapted this technique to give a complete strategy for direct sequencing of PCR products that yields reproducible high-quality sequence data and uses the same primers as the PCR amplification. The method is fast (25 min), can purify several PCR products simultaneously and allows direct sequencing of products up to 500 bp. Standard PCR protocols can be used, and both radioactively or fluorescently labeled nucleotides are suitable for use during sequencing.