Human-Specific DNA Quantitation

DNA purification methods cannot differentiate between human DNA and other DNA (e.g., bacterial and fungal DNA). Therefore, if the sample is not pristine and you want to determine the concentration of human genomic DNA present, you will need to use a human-specific DNA quantitation system. In forensics, the most common systems are based on real-time PCR and harness fluorescently labeled oligonucleotide probes or primers to detect and quantitate a PCR product in real time. The progress of the reaction can be monitored as a change in fluorescence over time as specific PCR products accumulate. During the exponential phase of PCR, the change in fluorescence is proportional to the accumulation of PCR product. 

The PowerQuant™ System is a five-dye, four-target qPCR multiplex using hydrolysis probe technology. The system includes three multi-copy targets (autosomal, Y and degradation), an internal positive control (IPC) and a passive reference, each in a different dye channel. Amplification of the autosomal target determines the total human DNA concentration, and the Y amplification determines the human male DNA concentration in an extracted sample. The degradation target evaluates the degree of DNA degradation. The IPC target is an internal control for the amplification reaction and assesses whether or not PCR inhibitors are present. The recommended protocol for the PowerQuant™ System uses a four-point standard curve created from serial dilution of the PowerQuant™ Male gDNA Standard. DNA quantification using the PowerQuant™ System provides rapid results with a thermal cycling protocol on the Applied Biosystems® 7500 Real-Time PCR System that requires approximately 1 hour.

The Plexor® HY System is a real-time PCR assay that also determines the concentration of total human DNA and male human DNA simultaneously in one reaction. The kit contains an internal PCR control to test for false-negative results that may occur in the presence of PCR inhibitors and a melt curve function to confirm that the correct product was amplified. The Plexor® HY System measures reduction in fluorescent signal during amplification. Amplification of each target uses only two primers, one of which contains both a fluorescent tag and a modified base. As amplification proceeds, fluorescence is reduced by site-specific incorporation of a fluorescent quencher opposite the complementary modified base. The quencher is in close proximity to a fluorescent dye located on the end of the primer, resulting in a reduction of fluorescent signal. After PCR, a melt analysis can be performed to provide an internal control for the final assay design or to expedite troubleshooting.