Improved Nucleic Acid Recovery From Trace and Degraded Samples Using Affinity Purification

Collection of trace and environmentally damaged DNA using traditional methods, such as ion exchange and silica beads, remains challenging. This is due to 50-90% losses associated with limited input sample material and low binding affinity of short DNA fragments to the silica beads. We propose to develop capture agents that provide higher initial recovery of trace DNA. In addition, the molecular biology based forensic science toolkit is expanding beyond sole reliance on DNA analysis. Recently, RNA from forensic material has been used to discern the tissue or bodily fluid source and postmortem interval based on RNA degradation half-life, and these techniques are anticipated to gain wider acceptance over the coming years. Emerging methods for evaluating protein polymorphisms and metabolites may be complementary to conventional DNA analysis in cases where the DNA evidence is inconclusive. However, the ability to recover all of these molecules depends on the development of new non-destructive approaches.  

Current commercial sample prep kits typically isolate either DNA, RNA, or protein and use chemicals that are destructive to recovery of the other molecules. We propose to develop a novel method to improve trace and degraded DNA purification that also allows for differential RNA binding and elution and is non-destructive to proteins and metabolites in the sample. Our preliminary data shows binding and elution of DNA sizes ranging from >10 kb to ~25 bp, with recoveries of up to 95% of the initial DNA amount. Therefore, even highly degraded DNA samples could be recovered using this approach.

Protocols for differential DNA and RNA binding and elution will be developed that are effective in common interferants present in forensic samples, and the workflows will be refined on mock forensic samples including blood, saliva, and shed skin. The impact of environmental damage will be evaluated. In addition, swabs modified with the nucleic acid capture agent will be developed for direct nucleic acid recovery from the evidentiary sample. To demonstrate compatibility with downstream human identification methods, recovered nucleic acids will undergo STR analysis, RNA-Seq, and/or RT-PCR.

Our proposed approach is amenable to automation, would benefit trace DNA evaluation, and could enable a future workflow in which trace forensic samples can be analyzed for a full suite of diverse molecular content, leading to improved accuracy of criminal investigations. Success in this project will lay the groundwork for future development of commercial kits and precision approaches that address specific forensic workflows.