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Modified PCR amplification and direct sequencing procedures for the double-stranded genomic DNA template are described. Advantages of the approach we describe are: background artifact bands previously observed using high-molecular-weight DNA as a template were eliminated by this protocol; no gel purification or subcloning of the PCR-amplified double-stranded fragment was required prior to direct sequencing; and sequences of 300 nucleotides can be easily read even after a single loading. The successful use of the modified dideoxynucleotide chain-termination method for direct sequencing of both strands demonstrates the efficiency of this technique for removing sequencing artifacts and for producing reliable sequence data.

Reliable and efficient direct sequencing of PCR-amplified double-stranded genomic DNA template.