Brian Davis*, Michael Rishel, Wei Gao, John Nelson, and Chrystal Chadwick | GE Research Coral Smith and Arati Iyengar | West Virginia University Abstract: An increasing proportion of DNA casework in the United States comprises trace levels of DNA. Traditional kits for nucleic acid recovery present potential for loss of DNA during extraction and purification caused by multiple wash steps and buffer exchanges during which the small amount of DNA can be lost. Although this loss may be insignificant on body fluids such as blood or semen where DNA content is high, when working with trace DNA, it could mean the difference between an informative profile and unusable data. In addition, commercial kits for DNA recovery disregard the potential value of RNA, proteins, and metabolites and focus exclusively on DNA isolation. RNA and metabolites are highly valuable because they can identify the type of biological sample within the evidentiary sample. There is an immediate need for an efficient workflow that effectively extracts and purifies DNA from challenging trace samples while simultaneously preserving RNA, proteins, and metabolites for further analysis. The researchers’ new approach combines high- efficiency recovery of trace DNA with retention of proteins and metabolites in the unbound material. The significance of additional analytes has been demonstrated in recent literature; however, adaptation of these techniques has been delayed because of a lack of an efficient multianalyte isolation kit. This research seeks to fill that gap by developing a novel workflow incorporating a highly efficient DNA binder imbedded into a paramagnetic bead in combination with buffers that are non-destructive to protein and other analytes. Preliminary data using known quantities of DNA applied to the DNA binder and from swabs of glass slides handled by volunteers (with/without spiked DNA) demonstrated near complete binding of nanogram quantities of sheared (size range 500 bp to 12 kb) human genomic DNA, while the remaining analytes are retained in the unbound material for further analysis. Current elution protocols achieve up to 90% recovery from the DNA binding vector, confirmed by the Quant-iT™ PicoGreen™and Quantifiler™ Trio assays (Thermo Scientific™). With DNA of 25 bp to 500 bp, 95% elution efficiency was obtained, indicating usefulness of this method with severely degraded DNA that is often problematic with conventional kits using DNA binding columns. Experimentation is in progress to improve DNA elution efficiency through enzymatic release agents and buffer optimization and to develop optimal protocols for co-binding and differential elution of RNA from the binding vector. Results will be presented on DNA elution efficiency using enzymatic release agents and several buffer combinations and on different strategies for co-binding and differential elution of RNA. Side-by-side comparisons to currently available commercial kits will also be presented. In time, this innovative research will result in the development of a robust method of successfully isolating challenging trace DNA while retaining RNA, protein, and metabolites from the same sample, thus increasing the value of a single piece of biological evidence.