Dideoxy Sequencing Reactions Using Taq Polymerase

Tuq DNA polymerase is a highly stable polymerase isolated from the thermophilic organism Thermus aquaticus . Because of its unique properties this enzyme has been extensively used for amplification of DNA fragments by the polymerase chain reaction (PCR). In addition, the thermostability of Tuq DNA Polymerase has proven to be extremely useful for sequencing single-stranded templates by the dideoxy chain termination reaction (1 –3 ). The advantage of sequencing with Tuq DNA polymerase is that the reaction can be extended at high temperature, thus eliminating most band compression encountered when sequencing G + C-rich templates or hairpin regions. This method is particularly useful when sequencing DNA from organisms with a high G + C content where ambiguities are frequently found on the gels (see Fig.1 ). Currently several companies have developed sequencing kits using Tuq DNA polymerase. We have tried the TAQence^TM (U.S. Biochemicals, Cleveland, OH) and the Tuq multiwell^TM (Amersham, Arlington Heights, IL) kits, both of which gave comparably good results. However these kits are relatively expensive and similar results can be obtained by preparing the different solutions with little loss of time. Here we describe a protocol based on the chain termination reaction as described by Stambaugh and Blakesley (4 ) that involves two major steps.

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