Reliable and efficient direct sequencing of PCR-amplified double-stranded genomic DNA template.
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.
Jung
M
M
Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007.
Dritschilo
A
A
Kasid
U
U
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000235:genetics; D005069:Evaluation Studies as Topic; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D015342:DNA Probes; D004273:DNA, Neoplasm; D004247:DNA