By Michael M. Cox, Ph.D., Evelyn M. Mercer
Published by the NIJ
Abstract
Almost every day, DNA samples are collected from the scenes of crimes or disasters that are too degraded for standard forensic DNA analytical procedures. This fact represents an ongoing impediment to law enforcement and victim identification efforts. Many law enforcement agencies also possess archived crime scene evidence from cold cases that are decades old, in which the DNA has become too damaged to analyze. The major problem in these samples is the presence of DNA double strand breaks. The purpose of the work carried out under grant 2010-DN-BX-K190 is to develop a new method to repair double strand breaks in forensic DNA samples, as a pretreatment for the standard STR analysis protocols. As part of this effort, we have also developed reproducible procedures for the artificial degradation of human DNA samples, using ionizing radiation to inflict a DNA damage profile that reprises that of a typical degraded forensic sample. Using this type of DNA as a test bed, we have developed a protocol that is successful in increasing/restoring missing or substandard signals at two STR loci. The protocol utilizes the bacterial RecA protein, single-stranded DNA binding protein (SSB), and bacterial DNA polymerase I, in concert with a targeting oligonucleotide. The reactions promoted by these reagents effectively restore damaged DNA flanking a particular STR locus. With the most developed protocol, signal restoration is successful approximately 20% of the time. With a few exceptions, the restored signals are accurate. The artifacts arising in the exceptions have been traced to the targeting oligonucleotides. Efforts to further develop this technology are continuing, focused on new RecA protein variants that increase signal strength and re-designed targeting oligonucleotides.
We have also been successful in demonstrating proof of principle in efforts to recover targeted DNA segments and remove them from bulk DNA in an effort to concentrate them and eliminate conditions that could inhibit STR amplification.
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