Identification of a crime perpetrator on the basis of DNA fingerprinting is not as easy as some of the CSI shows on television make it out to be. A sample such as blood stain, touch sample or body fluid retrieved at a crime scene is often a challenge for DNA analysts. In many instances, the samples are limited in quantity, found in dirty conditions, exposed to harsh environmental factors and are mixtures of more than one DNA—human and/or non-human. One of the most important aspects of the workflow to successfully obtain a DNA fingerprint profile is accurate quantification of human amplifiable DNA. The more information gleaned from the sample, the better equipped the DNA analyst is to determine the best course of action for obtaining a usable short tandem repeat (STR) profile from challenging samples. Therefore, Promega has developed the PowerQuant™ System, a probe-based 4-target, 5-dye real-time PCR method to a) determine human and male DNA concentrations in a sample, b) detect possible PCR inhibitors c) identify possible mixtures and d) measure DNA integrity. This DNA quantitation system offers the following benefits:
Sensitivity: Before STR analysis, it is important to know if there is human amplifiable DNA in the sample. If no DNA is detected, (assuming the DNA quantification assay is sensitive enough), the DNA analyst can be confident about not proceeding to STR analysis with samples with 0 quantitation values, thus saving time and resources. For this reason, an ideal DNA quantification kit must be extremely sensitive. If DNA is detected, it is important to determine the concentration accurately because STR kits are optimized for a narrow range of DNA input for a balanced profile output. Incorrect DNA quantification would cause the DNA analyst to add too little or too much DNA, leading to missing or sub-threshold peaks or overblown peaks in the subsequent STR profile and often forcing the analyst to repeat the assay. This wastes time and reagents. The PowerQuant™ System amplifies a multi-copy autosomal target (similar to that for the Plexor® HY System) with sub-picomolar sensitivity, ensuring that no DNA detected in quantification step translates as no usable STR profile.
Mixture: A large percentage of casework samples, such as sexual assault case samples, contain high female-low male DNA mixtures, and therefore, it is important to determine if an autosomal or Y-STR would be the optimal downstream processing path. The PowerQuant™ System amplifies autosomal and Y chromosomal targets simultaneously and allows calculation of the Auto/Y DNA concentration ratio to determine the ratio of male and female components in the samples.
DNA quality: Apart from knowing the DNA concentration, it is also useful to estimate DNA quality—for instance, to detect possible PCR inhibitors in the sample or determine DNA integrity. Humic acid, hematin and tannic acid are general PCR inhibitors frequently encountered in casework samples originating from soil, blood and leather fabric. The presence of PCR inhibitors can cause imbalance or loss of peaks at certain loci and may generate an incomplete profile. Human DNA quantification kits, including PowerQuant™ System, contain an Internal PCR Control (IPC) template along with IPC primers in every reaction. The presence of PCR inhibitors in the sample negatively affects IPC amplification, leading to later Cq values as compared to IPC Cq values of DNA standards or controls. This shift in IPC Cq values enables the DNA analyst to make informed decisions about DNA quality prior to STR analysis.
If the casework sample has been exposed to harsh environmental conditions, the DNA may be degraded and fragmented. Because there are fewer longer template DNA molecules, larger amplicons may show smaller or absent peaks after STR amplification. This results in a “ski slope” profile, where the peak heights decrease progressively with an increase in amplicon size. To estimate DNA quality prior to STR amplification, the PowerQuant™ System amplifies short (Auto) and long (Deg) autosomal targets from the same sample simultaneously. If the DNA integrity is good, the larger amplicon will be amplified with approximately the same efficiency as the smaller autosomal amplicon, and the ratio of the quantitation values (Auto/ Deg) will be close to 1. Conversely, if the DNA is degraded, there are fewer starting template molecules that span the larger amplicon and so, the larger autosomal target shows a later Cq and the Auto/ Deg ratio is higher than 1. Determining the DNA quality prior to STR amplification again allows the DNA analyst to make informed choices about processing challenging samples.
In summary, the PowerQuant™ System is a robust real-time PCR quantification kit that provides qualitative and quantitative information about forensic casework samples in about an hour to predict the success of obtaining a STR profile.
Anupama Gopalakrishnan
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