Despite recent advancements, fully automated rapid PCR instrument analysis still takes 90 minutes. While effective on high-quality and high quantity samples such as known references, complete DNA profiles are difficult to obtain from challenging samples such as low-level saliva, blood and semen. Lack of sensitivity is the main issue when processing these challenging forensic samples for rapid direct DNA analysis.
Since microwaves can disrupt cell membranes, their use prior to DNA analysis can provide a quick and inexpensive method to augment the recovery of DNA prior to rapid Direct PCR (no extraction). The hypothesis of this study is that microwaving low levels of blood, semen and saliva can increase DNA yield and profile recovery without requiring any further extraction.
This project is the first of its kind to use microwave treatment as an extraction step for DNA samples as a front end to rapid direct PCR. The project consisted of three main goals: 1) develop a fast, inexpensive, efficient microwave-based extraction technique as front end for direct PCR 2) optimize an in-house rapid direct miniSTR multiplex for speed and sensitivity and 3) validate the methodology through an interlaboratory study.
Results from microwave calibration and testing demonstrated the highest DNA yield was achieved using 300W power setting for a total of 40 seconds run time. An in-house multiplex of 9 mini STRs was developed and amplified samples in less than 15 minutes from all the three body fluids using no extraction steps other than the microwave treatment.
Microwaving samples prior to conventional capillary electrophoresis DNA analysis resulted in a 340% increase in allele recovery from 1% blood dilutions, 55% increased recovery from 1% saliva dilutions, and 96% increased recovery from 0.5% semen dilutions. In 1:9 saliva/semen mixtures, the total number of detected alleles increased from 20 to 25 alleles with microwaving, with the minor donor allele peak heights increasing 106%