Rescue of highly degraded DNA by primerless PCR
Next-generation sequencing (NGS) platforms are now implemented as routine analysis for K-RAS mutation in colon cancer patients before therapy. The DNA used in NGS platforms is extracted from colon cancer formalin-fixed paraffin-embedded (FFPE) blocks. In this study, we utilized 20 FFPE colon cancer blocks. In general, a good quality DNA sample includes compact high molecular weight DNA. The quality of the extracted DNA is checked by agarose gel electrophoresis. Some samples are found to be highly degraded due to natural mechanisms like autolysis and spontaneous depurination, or bacterial contamination and extracted DNA is then routinely fragmented by sonication. In this study, PCR was performed to reconstruct larger DNA fragments rather than to amplify DNA fragments. Primerless PCR relies on the natural power of two segments of the PCR cycle to reconstruct fragmented PCR by: the capability of denatured DNA to anneal randomly to its complementary sequence (annealing), and Taq polymerase to extend the DNA at the 3’ end (extension). By repeating for 150 cycles a larger DNA fragment is generated instead of amplifying the DNA. Fragmented DNA was reconstructed by primerless PCR for 150 cycles. However, 1U of Taq polymerase was added to the PCR reaction every 50 cycles. The samples selected for this study were highly degraded. The degree of degradation of samples was visualized by running the samples on a 1% agarose gel. After running primerless PCR for 50, 100, and 150 cycles, larger fragments of highly intact DNA appeared as sharp, compact bands whereas degraded DNA was a diffuse smear. In conclusion, we have demonstrated that primerless PCR can authentically generate DNA fragments that are larger than the initial template, and the DNA polymerization of self-primed DNA fragments can increase the likelihood of successful regeneration ostensibly by reconstructing the template. Primerless PCR can help in regenerating larger DNA fragments from highly deteriorated samples, such as forensic, and ancient samples.