Tools for Improving the Quality of Aged, Degraded, Damaged, or Otherwise Compromised DNA Evidence
John R. Battista
Abstract
Current forensic DNA genotyping technology requires 0.2-2.0 nanograms duplex DNA that is at least 100-500 base pair in length. However, many evidence samples fail to meet these minimal requirements, because the target DNA has been exposed to environments capable of extensively damaging DNA. This damage reduces the size of the target DNA, making it too small to be amplified and can block PCR amplification. Since DNA samples found at crime scenes exhibit varying degrees of damage, the effectiveness of DNA typing technology is limited by sample condition, and at present there are no methods of circumventing this problem. Our principle objective was to develop tools to facilitate STR DNA genotyping through a) improving the quality of the DNA found in degraded forensic samples, and b) enhancing the ability to retrieve amplifiable DNA from forensic samples. Our efforts were focused on two areas. First, we attempted repairing damaged DNA in vitro using cell extracts isolated from repair proficient microorganisms. Extracts were emphasized because there is no way of knowing a priori what type of DNA damage is present in biological evidence, and intact cells express an extensive array of DNA repair proteins capable of dealing with most DNA damage. These efforts met with minimal success; it appears we were able to repair DNA in vitro, but the technique was difficult to reproduce. The cell extracts inhibited the reaction used to monitor repair and prevent an accurate assessment of the extent of repair. Second, we have developed a method for retrieving selected DNA fragments from complex mixtures. DNA damage occurs randomly and so the double strand breaks resulting from that damage are also random. A sample – even a highly degraded sample – is expected to have a distribution of fragment sizes, and our efforts are aimed at isolating fragments that include the intact STR sequences from such mixtures. To that end we have developed an in vitro method using three purified proteins that permits selective capture of specific DNA sequences. This method relies on the use of a targeting DNA fragment, a sequence that is complementary to the sequence you wish to retrieve. Combining this fragment, the three proteins, and appropriate cofactors allows recovery of homologous DNA from solution with high efficiency.