One step DNA sequencing of single-stranded DNA with reverse dye-primer.

DNA sequencing can be performed much faster and more easily by an automated DNA sequencer, which can process a large quantity of samples at one time. We use an Applied Biosystems DNA sequencer, Model 373A. Among several methods applicable to the sequencer, the dye-primer method of singlestranded template with Taq DNA polymerase gives the most reliable results. If both dye-primers (forward dye-primer (-21M13) and reverse dye-primer (M13RP1)) are available, the dye-primer method will be able to give at least 400 nucleotide sequences from each end. However, only the forward dye-primer is available for a single-stranded template. For use of a reverse dye-primer, two methods are available. 1) The insert can be recloned into another vector. 2) A double-stranded fragment can be produced by PCR with both primers. In both cases, an additional step is required, providing a large obstacle to extensive DNA sequencing.