A rapid polymerase-chain-reaction-directed sequencing strategy using a thermostable DNA polymerase from Thermus flavus.

We have developed a polymerase chain reaction (PCR)-directed sequencing strategy for rapid sequencing of DNA from crude viral or cell preparations. The basic strategy consists of two phases. In the first phase, the target DNA is amplified by symmetric PCR with low concentrations of deoxyribonucleotide triphosphate (dNTP) and oligodeoxyribonucleotide primers. This results in exponential amplification of DNA in the initial cycles, reaching a plateau by 25 cycles due to limiting concentrations of dNTP and primers. In the second phase, a small aliquot of the PCR mixture is amplified without any purification, by asymmetric PCR in the presence of a 5'-labeled primer and one of the four dideoxyribonucleotide triphosphates. This results in the accumulation of single-stranded DNA products that are terminated at specific points by incorporation of the appropriate dideoxyribonucleotide monophosphate. The products are then analyzed by electrophoresis on a sequencing gel followed by autoradiography. The PCR conditions are optimized to generate sequence ladders of several hundred nucleotides starting from as low as 100 copies of bacteriophage or bacterial genome in one to two days.