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Mis-priming of DNA template is one of the most common problems associated with the dideoxy chain termination method of DNA sequencing (1). It can be a particular problem with GCrich primers and templates, or with templates containing repeats. Careful primer design with the use of primer design software can alleviate many of these problems when sequencing known sequences but is not possible with unknown sequences. Taq sequencing is gaining popularity and should not, in theory, suffer from the problems of mis-priming, but there are many who believe it has yet to be optimised and who prefer more traditional protocols. Mis-priming may be partially overcome by the use of glycerol-tolerant sequencing gels (2), allowing sequencing extension reactions to be carried out in the presence of high concentrations of glycerol and thus at higher temperatures. We have found that a simple stringency wash can eliminate mispriming completely when using the Dynabeads/biotin system (Dynal Ltd, Merseyside, UK) for the generation of singlestranded DNA template (3).

A method for avoiding mis-priming when sequencing with Dynabeads