A quantifiler™ trio-based HRM screening assay for the accurate prediction of single source versus mixed biological samples.

At present, the forensic DNA workflow is not capable of providing information about the contributor status (single source vs. multiple contributors) of evidentiary samples prior to end-point analysis. This exacerbates the challenges inherent to mixtures and low-template DNA samples. If additional sample information could be provided earlier in the workflow, protocols could be implemented to mitigate these challenges. An integrated Quantiplex®- high resolution melt (HRM) assay was shown to be effective in distinguishing between single source and mixture DNA samples; however, integration of the HRM assay into a more commonly used chemistry would be beneficial to the practitioner community. Thus, the assay was redesigned as an integrated Quantifiler™ Trio-HRM assay, which included the identification of a new DNA-binding dye, an increased reaction volume, and the establishment of new data analysis and standard curve metrics for all targets. This redesigned assay produced quantification values and qualitative values that were comparable to those produced when the same samples were tested using the standard Quantifiler™ Trio chemistry and settings. Further, STR profiles generated with quantification values produced from the integrated Quantifiler™ Trio-HRM assay and standard Quantifiler™ Trio chemistry were complete and fully concordant. Most importantly, the integrated Quantifiler™ Trio-HRM assay was able to accurately predict whether a sample was single source or a mixture 79.2% of the time, demonstrating the potential of this approach. With the incorporation of an expanded training set for prediction modeling, and completion of critical developmental validation studies, this assay could prove useful to the forensic DNA practitioner community.

Yield Gel via Quantitative Gel Electrophoresis.

Quantitative gel electrophoresis, also referred to as yield gel via gel electrophoresis, is an early quantification method that was developed to provide an estimate of the quality and the quantity of DNA extracted from evidence or reference samples. To conduct quantitative gel electrophoresis, an agarose gel that is combined with a nucleic acid gel stain is prepared. The gel stain intercalates between double-stranded DNA and can be visualized using UV light. DNA extract samples, along with DNA standards (ranging from 250 to 5 ng), and a 1 KB ladder are combined with a 6X loading dye and loaded on the agarose gel. Voltage is applied to facilitate DNA migration through the gel from the negative to the positive electrode, separating DNA fragments by size. After electrophoresis is complete, the results are visualized using UV light, and an image is captured for analysis. High-quality and -quantity DNA should contain a compact band comparable to that of the high molecular weight standards and ladder, with some smearing down the sample well. If a DNA extract sample does not produce a compact band and presents with only a smear, this is an indication that DNA degradation has occurred. This chapter provides instructions on how to successfully prepare an agarose gel, load DNA extract samples and corresponding controls, appropriately set up and run quantitative gel electrophoresis, interpret the results, and ensure comprehension of the method so troubleshooting can be performed if needed.