A number of sequencing reaction parameters are critical to success. First, increased amounts of the amplified yeast template tend to elevate background detected in the autoradiograms. In addition, a heterogeneous amplification product resulting from the initial amplification also may produce high backgrounds. If this situation arises, sequencing must be performed following gel purification of the amplified yeast sequences. We also do not recommend use of the initial amplification primers as the end-labeled sequencing primer without gel purification of the amplified yeast sequences.
Unlabeled primers compete in the sequencing reaction resulting in decreased signal.
Figure 2 presents the sequencing results of two yeast a -factor mutants using this method and demonstrates the quality of the sequence data obtained using the described procedure. Following the above protocol on a homogeneous amplification product, the quality of sequence data is excellent and can be obtained in a single day. Results from two yeast mutants using the rapid direct sequencing method. Summary Direct sequencing of plasmid DNA from yeast transformants using this method allows for rapid screening at the sequence level. The complete time for such a procedure has been decreased from several days to one day, and the method is now amenable to processing of many samples simultaneously.