Reliable and efficient direct sequencing of PCR-amplified double-stranded genomic DNA template.
Open Access
- 1 February 1992
- journal article
- Published by Cold Spring Harbor Laboratory in Genome Research
- Vol. 1 (3) , 171-174
- https://doi.org/10.1101/gr.1.3.171
Abstract
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.Keywords
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