Meeting Abstract
There are many situations where investigators are faced with degraded DNA samples, but still need to obtain sequence data. This can include the analysis of ancient DNA, museum specimens, and even fresh tissues that have been delayed in transit and allowed to decompose. Particularly when samples are rare or prohibitively expensive to replace, it can be important to maximize data obtained from limited and damaged material. Two major problems are low template concentration and fragmented template. We explored the efficacy of touchdown and nested PCR strategies, without the use of special reagents, to recover sequences under less than ideal situations. We found that when DNA quantities are very low, it is difficult to amplify and visualize the data. We used the touchdown PCR technique to minimize the use of template DNA and avoid temperature optimization for each primer/template combination. Theoretically, PCR can work with a single strand of template, however, starting with very low concentration typically does not yield enough product to obtain reliable sequence information. We used the nested PCR technique, adding a second set of primers that are designed to sit internally to the original primer set. We were able to use the PCR product from the initial touchdown PCR as template for the nested PCR, yielding high concentrations of amplified DNA to visualize on agarose gels and Sanger sequence. Using combinations of these techniques we were able to obtain sequences of up to 600 base pairs for phylogenetic study, even in samples with little high molecular weight template (too low to visualize on an agarose gel). These methods may be applicable to many situations where template is degraded and in low quantity.
