Development of a Forensic DNA Quantification Assay using Digital PCR

Quantification of DNA in extracted samples offers key insights for an examiner’s decision on which downstream analysis to conduct. If the amount of nuclear DNA in a sample is too low for autosomal STR analysis, there may be the option to amplify the mitochondrial DNA present in the sample. Currently, however, these quantifications are achieved in two separate qPCR assays which increases the cost, time for analysis, and sample consumption. Additionally, the use of qPCR introduces the risk of imprecise measurements due to the relative quantification of samples compared to the standards, which themselves may also introduce some level of imprecision. A newer technology, digital PCR, eliminates the need for standards by separating a PCR reaction into thousands of nanoliter reactions to provide direct, absolute quantification.

The concept of dPCR is to take a standard volume PCR quantification reaction and to divide it into thousands of partitions to create independent PCR reactions. These nanoliter-sized reactions still contain all the components necessary for PCR, but commonly with only one copy of the target DNA in the partition. With a known number of total partitions and the ability to count individual partitions fluorescing at a specific wavelength, the number of copies of target DNA in the original sample can be determined directly using Poisson statistics, but without the need for standards. This technology also boasts a lower limit of quantification. The latest versions of these instruments can query at least five different dye channels, providing an opportunity to simultaneously probe the concentration of nuclear, mitochondrial, and male DNA, as well as a target for degradation and an internal positive control for inhibition.

In this presentation, the initial development for this combined nuclear and mitochondrial DNA quantification assay will be discussed. Efforts thus far have focused on translating the Federal Bureau of Investigation’s mitochondrial qPCR assay to the QIAGEN QIAcuity dPCR instrument. The work conducted has been: 1) to verify that qPCR and dPCR produce comparable results for the individual components of the mitochondrial quantification assay, 2) optimization of primer and probe concentrations for reproducibility, 3) identification of optimal fluorophores for improved signal strength, 4) testing common inhibitors (Humic Acid, Tannic Acid, Hematin, EDTA, Collagen, Melanin and Guanidine Thiocyanate) to determine if the mitochondrial quantification assay is more resilient to inhibition using dPCR, and 5) testing assay sensitivity to as few as 0.5 copies per microliter.

The results of the experiments thus far demonstrate an exciting new chapter for DNA quantification in forensics. With the migration of the mitochondrial quantification assay to dPCR and various modifications to the reaction mix, mitochondrial DNA can be quantified with comparable precision and accuracy to qPCR, but without the need for standards, providing some savings on time and costs. With the future incorporation of nuclear and male DNA targets to the assay, this quantification kit could allow forensic laboratories to increase the speed and accuracy of their measurements, consuming less volume of sample and, leading to improved workflows that deliver faster results for laboratory customers.