Direct Sequencing of Genomic DNA

Ryan Richt¹, Elaine Mardis², Biology Department, Washington University, St. Louis, MO^1, Department of Genetics, Washington University School of Medicine, Genome Sequencing Center
Direct Sequencing of Genomic DNA

Current protocols for determining the sequence of a specific gene from a bacterial genome, for example, require an intermediate PCR step to amplify the sequence of interest, followed by a DNA sequencing reaction. A protocol allowing us to bypass this intermediate PCR step would greatly decrease the expense and time required for these types of assay. I have shown that sequencing reactions using gene specific primers can be performed on total genomic DNA. A series of experiments confirms not only the feasibility of the method, but also begins to hint at optimized reaction conditions. Two micrograms of bacterial genomic DNA with 1 to 5 nanograms of primer are sufficient to yield read lengths of 560 quality bases. Such reactions perform optimally with 2 microliters of BigDye sequencing cocktail in a 10 microliter reaction volume. Cycling parameters differ from conventional plasmid sequencing only in the addition of an initial 5 minute denaturation at 95 C. While high copy genes produce the longest read lengths, even double copy genes are in sufficient quantities to yield over 425 base pairs. Single copy genes yield shorter read lengths of approximately 180 base pairs. Further optimizations could lead to improved sequence data from equal or less template, but even the described method could prove useful in both sequencing and diagnostic projects.