The effects of metal ion PCR inhibitors on results obtained with the Quantifiler® Human DNA Quantification Kit
Introduction
Forensic DNA testing laboratories across the globe employ multiplex short tandem repeat (STR) typing kits for human genetic identification and forensic casework. These analyses are conducted through the use of commercially-available kits, such as Identifiler® Plus (Applied Biosystems®, Foster City, CA) and the PowerPlex® 16HS System (Promega Corporation, Madison, WI). These kits are designed to co-amplify targets of 15 polymorphic STR loci plus Amelogenin, a sex determining marker, and contain advanced buffering components designed to produce results in the presence of polymerase chain reaction (PCR) inhibitors [1], [2]. Optimal performance of these kits requires adding DNA template to PCR reagents within the working range of the kit. If too little DNA is added to the reaction, it results in the preferential amplification of small loci, increased stochastic amplification, and a failure to obtain enough product to reach interpretation thresholds; conversely, too much DNA can lead to spectral overlap (pull-up) and incomplete adenylation [3]. These phenomena can cause interpretation challenges during analysis of multiplex STR data and may be avoided by adding a quantity of amplifiable DNA within the optimal working range of the kit. It is imperative to obtain an accurate quantification estimate of the DNA prior to STR amplification for optimal performance of these kits and ease of data interpretation.
There are several commercially-available chemistry systems that provide estimates of the amount of amplifiable DNA in a sample, most of which are designed for quantitative real-time PCR (qPCR)-based platforms. The qPCR-based quantification systems used by forensic DNA testing laboratories, such as the Plexor® qPCR System (Promega Corporation, Madison, WI), the Quantifiler® Human DNA Quantification Kit (Quantifiler kit; Applied Biosystems) and the Investigator® Quantiplex Kit (QIAGEN, Hilden, Germany), share conceptual similarities in their assay designs and physical kit components. Each system includes a human-specific probe that targets an evolutionarily conserved region of the human genome. While the physical design varies between manufacturers, these probes contain a fluorescent reporter dye whose fluorescence is altered as a consequence of target amplification and allow the amount of emitted fluorescent signal to be detected during each PCR cycle [4], [5], [6]. All qPCR-based quantification methods compare the fluorescent signal measured in a sample to a passive reference signal; this is a fluorescent component that does not change as a result of cycles of PCR. During absolute quantification, the cycle number of the PCR where there is a measureable difference between a sample’s fluorescent signal and the passive reference signal is recorded as the quantification cycle (Cq); absolute quantification estimates are derived by comparing a sample’s Cq to an external calibration curve from a dilution series of reference standards [7], [8], [9].
All qPCR assays are sensitive to factors that affect all types of PCR amplifications. Quantification results are estimates, not actual measurements, of the quantity of DNA in a sample and may not always reflect the true quantity present. Anything that changes the PCR conditions, such as the availability of reagents, pH or salt concentration, DNA polymerase function, or access to the DNA template, can impact the accuracy of the results [10]. Assessment of the individual quantification reactions and detection of substances that can inhibit PCR is aided by the inclusion of an internal PCR control (IPC) in each of the aforementioned quantification systems [4], [5], [6].
Early studies aiming to identify the presence of a PCR inhibitor in a DNA sample were conducted using spiking experiments by either adding the DNA sample to the positive control reaction or by adding the positive control sample to the reaction containing the DNA sample. In either case, failure to obtain amplification results from the positive control sample would serve as confirmation of the presence of an inhibitory compound in the DNA sample [11], [12]. The inclusion of IPCs into qPCR chemistry kits offers the same benefits provided by a positive control sample, evaluating reagent quality and instrument performance, as well as the possibility of identifying the presence of PCR inhibitors in a single, standardized reaction. The developmental validation studies and user manuals for these systems state that a negative IPC result with a negative human result is evidence of PCR inhibition and recommends treating the sample with dilution or additional purification; however, a positive IPC result with a negative human result is presented as “no human DNA detected,” “confirmation of negative results,” and “something that should instill confidence in the quantification results” [4], [5], [9].
Problems associated with the use of PCR-based assays for inhibitor detection and reliable quantification of inhibited samples have been presented in the scientific literature. It has been demonstrated that the effect of inhibitors on different PCR reactions is variable and there is no correlation between amplicon characteristics and the extent of inhibition, making it imperative to match the susceptibility to inhibitors between reference and experimental reactions to increase the accuracy and reduce the error of PCR-based quantification assays [13]. The potential of inhibitors to confound quantitative assessments of DNA quantity and increase the uncertainty of the values obtained has also been extensively studied [14]. Recognition of these issues has led to the development of statistical methods that examine amplification efficiency in order to improve quantification estimates and the detection of the presence of inhibitors, including identification of kinetic outliers and multivariate analysis of the dynamic phase of amplification between samples and controls [15], [16], [17]. The developers of these methods acknowledge that certain types of inhibition may not be detected, even using these advanced mathematical analyses.
The IPC will detect a PCR inhibitor, assuming the inhibitor blocks requisite reagents, inactivates or interferes with the processivity of the polymerase, and in some cases, binds to the DNA template [18]. In order for this inhibitor-DNA complex to be detected by an IPC, there must be sufficient inhibitor in the reaction to affect the IPC template and the sample template. Metal ions have the potential to function as inhibitors of PCR-based STR assays and are present in a variety of forensically-relevant sample types, e.g., bones [19]. Furthermore, metal ions can form soluble, high molecular weight complexes with humic substances in soils, have been implicated in adduct-formation with DNA, and can form crosslinks between DNA and proteins [20], [21], [22], [23]. These interactions can reduce the efficiency of DNA extraction techniques and impair access to the DNA template during PCR. Metal ions, including calcium, can also competitively inhibit DNA polymerases [18], [24]. If metal ion-template interactions prevent the DNA from being retained during extraction or are co-isolated as a complex, it is unlikely the inhibitor would be detected by an IPC assay because the inhibitor would not be available to interact with the IPC template. Due to the presence of metal ions in forensic samples and interactions with DNA that can reduce their detection by an IPC, it is important to elucidate their effects on qPCR assays.
The purpose of the following studies was to evaluate the ability of the Quantifiler kit to accurately estimate the concentration of amplifiable DNA in metal-treated DNA samples. Taking into consideration the potential challenges of attempting to quantify inhibited DNA samples, two questions are addressed: (1) Are DNA samples that include metal ions accurately quantified? and, (2) Does the IPC detect the presence of metal ion PCR inhibitors? Because metal ions have the potential to interact directly with DNA and inhibit PCR, it is important to elucidate the reliability of results obtained from PCR-based methods in their presence and the ability of the IPC to differentiate a true negative from an inhibited sample.
Section snippets
DNA sample preparation
Samples were prepared using solutions of purified aluminum (Al), calcium (Ca), copper (Cu), iron (Fe), nickel (Ni), and lead (Pb) obtained in the form of certified analytical standards (High-Purity Standards, Charleston, SC). An aliquot of each standard was diluted to 21 mM and the pH adjusted (between 3.8 and 5.8) using 3 M NH4OH and 1 M HCl. Dilutions of the metal stock solutions were created using UltraPure™ DNase/RNase-Free Distilled Water (Invitrogen, Carlsbad, CA). Experimentally-treated DNA
Estimated concentration of amplifiable DNA in metal-treated DNA samples
The standard curve parameters for each of the plates used in this study are listed in Table 2. The slope values are consistent with the typical range (−3.3 to −2.9) reported by the manufacturer [8], except for the Pb plate where the slope exceeded the lower-boundary of its range; however, this value was within the typical range observed in the laboratory. The intercepts reflect the expected Cq values for a 1 ng/μL DNA sample. These R2 values indicate a close fit between the standard curve
Conclusions and discussion
This study addressed two aspects of PCR-based DNA quantification using the Quantifiler kit, the accuracy of estimated DNA concentration in metal-treated samples and ability of the IPC detector to indicate the presence of metal ion PCR inhibitors. Comparing the estimated DNA concentration in metal-treated DNA samples with values obtained for positive control samples that were prepared with the same DNA concentration, conclusions can be drawn with respect to the accuracy of estimates for each of
Acknowledgements
Laura Gaydosh Combs is supported by the NIJ Ph.D. Graduate Research Fellowship Program FY 2012, award number 2012-IJ-CX-0017. DNA testing was performed with support from the National Institute of Justice program Using DNA Technology to Identify the Missing, FY 2010, award number 2010-DN-BX-K206. Vivian Huynh is supported by the NIJ Applied Research and Development in Forensic Science for Criminal Justice Purposes program FY 2013, award number 2013-R2-CX-K007. Joanna Castaneda’s work was
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