Development of a human-specific real-time PCR assay for the simultaneous quantitation of total genomic and male DNA

A duplex real-time quantitative PCR assay was developed for forensic DNA analysis, which provides simultaneous quantitation of total genomic human DNA and human male DNA. The assay utilizes two spectrally resolved fluorogenic probes in a 5' nuclease (TaqMantrade mark) assay. Within the range of organisms empirically tested and based upon theoretical specificity using National Center for Biotechnology Information GenBank sequences, primer and probe sequences were shown to be human specific, and the Y-chromosome probe, male-specific. A mixture-challenge study resulted in accurate quantitation of 25 pg male DNA in a mixture of up to 1:5000 (male:female DNA). Additional experimental results include comparisons with the slot blot method and commercial real-time PCR kits. The assay developed addresses the shortcomings of the traditional slot blot method as well as the commercial real-time PCR kits. This method is shown to be specific, relatively simple, rapid, has low limits of detection, and consumes limited sample in addition to reporting both the male and total genomic DNA concentrations present.     

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.     

Development of a real-time method to detect DNA degradation in forensic samples

Abstract: Knowledge of the degradation state of evidentiary DNA samples would allow selection of the appropriate analysis method (standard short tandem repeats [STRs] vs. mini STRs vs. mtDNA). This article describes the development of a Plexor® technology/real‐time PCR DNA degradation detection assay, which uses a common forward primer and two reverse primers (different fluorophores) to generate two Alu amplicons (63 and 246 bp). This very sensitive assay was optimized for reaction volume, cycle number, anneal/extend time, and temperature. Using DNA samples degraded with DNaseI, the ratio of the concentration of the short amplicon to the concentration of the long amplicon (degradation ratio) was increased versus time of degradation. Experiments were performed on a variety of environmentally degraded samples (age, sunlight, heat) and with seven commonly encountered forensic inhibitors. The degradation ratio was found to predict the observed loss of larger STR loci seen in the analysis of comprised samples.     

A real-time multiplex SNP melting assay to discriminate individuals

A method that quickly and inexpensively differentiates crime scene samples from multiple donors would expedite casework analysis by allowing the selection of probative items requiring comprehensive testing. This new method need not be perfectly definitive nor give a complete 13 locus short tandem repeat (STR) profile; it simply must be able to differentiate between most victim and suspect samples. We describe the development of multiplex, single nucleotide polymorphism (SNP), fluorescence resonance energy transfer-based real-time polymerase chain reaction (PCR) assays to fulfill this need. Dual probes, one fluorescently labeled and the other labeled with a quencher, are monitored during a melt analysis to reveal an increase in fluorescence, which allows the assessment of the two SNP alleles. Two alternate 6-plex assays (with and without gender determination) have been developed for the six-color RG6000 real-time instrument (Corbett Robotics, Inc.) and one seven SNP plus gender assay (performed as two 4-plex assays, one with gender the other without) have been developed for use in four/five color real-time instruments. This technique can discriminate between 95% and 99% of samples from different individuals. This assay is fast (approximately 2 h), much less expensive than STR analysis, and uses a real-time PCR instrument which is found in most forensic and molecular biology labs.     

Qiagen's Investigator™ Quantiplex Kit as a Predictor of STR Amplification Success from Low‐Yield DNA Samples,

Qiagen's Investigator? Quantiplex kit, a total human DNA quantitation kit, has a 200‐base pair internal control, fast cycling time, and scorpion molecules containing a covalently linked primer, probe, fluorophore, and quencher. The Investigator? Quantiplex kit was evaluated to investigate a value under which complete short tandem repeat (STR) failure was consistently obtained. Buccal swabs were extracted using the Qiagen QIAamp^® DNA Blood Mini Kit, quantified with the Investigator? Quantiplex kit using a tested half‐volume reaction, amplified with the ABI AmpFlSTR^® Identifiler kit, separated on the 3100Avant Genetic Analyzer, and data analyzed with GeneMapper^® ID v.3.2. While undetected samples were unlikely to produce sufficient data for statistical calculations or CODIS upload (2.00 alleles and 0.82 complete loci on average), data may be useful for exclusionary purposes. Thus, the Investigator? Quantiplex kit may be useful for predicting STR success. These findings are comparable with previously reported data from the Quantifiler? Human kit.     

STR Profiles from DNA Samples with "Undetected" or Low Quantifiler™ Results

Abstract:  Screening methods capable of identifying DNA samples that will not yield short tandem repeat (STR) profiles are desired. In the past, quantitation methods have not been sensitive enough for this purpose. In this study, low level DNA samples were used to assess whether Quantifiler™ has a minimum quantitation value below which STR profiles would consistently fail to be detected. Buccal swabs were obtained and the DNA extracted, quantified, and serially diluted to concentrations ranging from 0.002 to 0.250 ng/μL. Samples were analyzed once with Quantifiler™, followed by Profiler Plus™ amplification and capillary electrophoresis analysis. An absolute minimum value below which STR results were unobtainable could not be defined. From the 96 low level samples tested, STR loci (including one full profile) were successfully amplified and detected from 27% of the samples "undetected" by Quantifiler™. However, no STR alleles were detected in 73% of these "undetected" samples, indicating that Quantifiler™ data may be useful for predicting STR typing success.     

Developmental Validation of the Quantifiler® Duo DNA Quantification Kit for Simultaneous Quantification of Total Human and Human Male DNA and Detection of PCR Inhibitors in Biological Samples*

Abstract: The Quantifiler^® Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well. Pooled human male genomic DNA is used to generate standard curves for both human (ribonuclease P RNA component H1) and human male (sex determining region Y) specific targets. A shift in the cycle threshold (C_T) values for the internal positive control monitors the presence of PCR inhibitors in a sample. The assay is human specific and exhibits a high dynamic range from 0.023 to 50 ng/μL. In addition, the multiplex assay can detect as little as 25 pg/μL of human male DNA in the presence of a 1000-fold excess of human female DNA. The multiplex assay provides assessment of the DNA extract and guidance for the selection of the appropriate AmpFℓSTR^® Amplification Kit to obtain interpretable short tandem repeat profiles.     

Validation of a multiplex PCR assay for the forensic identification of Indian crocodiles

A dependable and efficient wildlife species identification system is essential for swift dispensation of the justice linking wildlife crimes. Development of molecular techniques is befitting the need of the time. The forensic laboratories often receive highly ill-treated samples for identification purposes, and thus, validation of any novel methodology is necessary for forensic usage. We validate a novel multiplex polymerase chain reaction assay, developed at this laboratory for the forensic identification of three Indian crocodiles, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, following the guidelines of Scientific Working Group on DNA Analysis Methods. The multiplex PCR was tested for its specificity, reproducibility, sensitivity, and stability. This study also includes the samples treated with various chemical substances and exposed to various environmental regimes. The result of this validation study promises this technique to be an efficient identification tool for Indian crocodiles and therefore is recommended for forensic purposes.     

Investigation of Reproducibility and Error Associated with qPCR Methods using Quantifiler® Duo DNA Quantification Kit*

Abstract: Reproducibility of quantitative PCR results is dependent on the generation of consistent calibration curves via accurate volume transfers and instrument performance. A review of 14 standard curves, using two different QuantDuo^® standard DNA lots, showed variability of cycle threshold values between assays were larger than those of the Internal PCR Control (IPC). This prompted a set of experiments designed to determine the source of variability. Results showed that error introduced during DNA addition to the plate resulted in little variation. A comparison of seven independent series demonstrated cycle threshold variation between dilutions was larger than the variation expected from repeated samples. Modeling the influence of pipette errors on dilution series accuracy indicated that a more rigorous approach to external calibration curve production is required and showed that improvement in calibration curve stability is expected if the pipette conditions are carefully chosen and/or a single validated curve is utilized as the calibrator.     

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability.     

An innovative transfer DNA experimental design and qPCR assay: Protocol and pilot study

Forensic “touch” DNA samples are low-quantity samples that are recovered from surfaces that have been touched by single or multiple individuals. These samples can include DNA from primary contributors who directly touched the surface, as well as secondary contributors whose DNA was transferred to the surface through an intermediary. It is difficult to determine the type of transfer, or how often and under what conditions DNA transfer occurs. In this paper, we present an innovative protocol that combines (1) a paired male and female transfer DNA experimental design in which the presence of male DNA indicates secondary transfer and (2) a cost-effective quantitative PCR (qPCR) assay of a sex-specific region in the Amelogenin gene to detect male and female DNA. We evaluate the ability of the Amelogenin qPCR assay to detect low concentrations of male and female DNA in mixed samples. We also test experimental DNA samples using our transfer DNA protocol to differentiate primary and secondary DNA transfer. Male DNA was detected in the majority of known mixed samples, even in samples with 4× more female DNA—this result demonstrates the ability to detect low concentrations of male DNA and the presence of secondary transfer DNA in our experimental design. Primary DNA transfer was detected in 100% of our experimental trials and secondary DNA transfer was detected in 37.5% of trials. Our innovative protocol mimics realistic case scenarios to establish rates of primary and secondary DNA transfer in an inexpensive and simplified manner.     

Modified Midi‐ and Mini‐Multiplex PCR Systems for Mitochondrial DNA Control Region Sequence Analysis in Degraded Samples

Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267 bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185 bp. Most mtDNA control region sequences of several 60‐year‐old and 400–500‐year‐old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis.     

Evaluation of real-time PCR amplification efficiencies to detect PCR inhibitors

Real-time PCR analysis is a sensitive template DNA quantitation strategy that has recently gained considerable attention in the forensic community. However, the utility of real-time PCR methods extends beyond quantitation and allows for simultaneous evaluation of template DNA extraction quality. This study presents a computational method that allows analysts to identify problematic samples with statistical reliability by comparing the amplification efficiencies of unknown template DNA samples with clean standards. In this study, assays with varying concentrations of tannic acid are used to evaluate and adjust sample-specific amplification efficiency calculation methods in order to optimize their inhibitor detection capabilities. Kinetic outlier detection and prediction boundaries are calculated to identify amplification efficiency outliers. Sample-specific amplification efficiencies calculated over a four-cycle interval starting at the threshold cycle can be used to detect reliably the presence of 0.4 ng of tannic acid in a 25 microL PCR reaction. This approach provides analysts with a precise measure of inhibition severity when template samples are compromised. Early detection of problematic samples allows analysts the opportunity to consider inhibitor mitigation strategies prior to genotype or DNA sequence analysis, thereby facilitating sample processing in high-throughput forensic operations.     

The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA

Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker((R)) 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the sensitivity of LCN DNA amplifications.     

The successful DNA typing of samples following a thermal cycler power loss

An approach for generating DNA profiles when critical samples have been consumed and a power outage occurs during the polymerase chain reaction (PCR) amplification reaction is described. This study demonstrates that a complete and accurate DNA short tandem repeat profile can be obtained: (1) when single source DNA samples are amplified for 26, 27, or 28 cycles using the Profiler Plus and COfiler Amplification Kits after an interruption in amplification, (2) from mock samples when PCR amplification has been interrupted early (after five cycles) or late (after 18 cycles) and the sample is subjected to an additional round of amplification, even after incubation of the sample at room temperature overnight, and (3) from nonprobative casework samples interrupted after approximately 18 cycles of amplification, an overnight incubation at room temperature and subjected to one or two additional rounds of PCR amplification for approximately 26 total cycles. Samples interrupted before five completed cycles and subjected to additional PCR cycles yielded variable results.     

An innovative transfer DNA experimental design and qPCR assay to identify primary,secondary, and tertiary DNA transfer

“Touch DNA” is a form of trace DNA that is presumed to be deposited when an individual touches something and leaves behind DNA-containing skin cells, sweat, or other fluids. While touch DNA is often the result of direct contact (i.e., primary transfer), it can also be indirectly transferred between surfaces or individuals (e.g., secondary or tertiary transfer). Even experts cannot distinguish between different types of transfer and do not fully understand which variables affect direct versus indirect transfer or how often each type of transfer occurs. In this study, we utilize an innovative protocol that combines a paired male and female transfer DNA experimental design with an Amelogenin qPCR assay to generate data on primary, secondary, and tertiary DNA transfer. We report frequencies of indirect DNA transfer and also investigate the potential effects of participant age, self-identified ethnicity, and skin conditions on DNA transfer. Out of 22 experimental trials, we detected primary transfer (male + female) in 71% of trials, secondary DNA transfer in 50% of trials, and tertiary DNA transfer in 27% of trials. No significant associations were found between primary DNA transfer and age, self-identified ancestry, or skin conditions, however, all individuals with sloughing skin conditions demonstrated primary DNA transfer and we suggest this variable be explored in larger samples. These results contribute to a better understanding of the conditions under which secondary and tertiary DNA transfer occurs and can be used to propose realistic DNA transfer scenarios in court cases.     

The effects of chemical and heat maceration techniques on the recovery of nuclear and mitochondrial DNA from bone

Forensic anthropologists use a number of maceration techniques to facilitate skeletal analysis of personal identity and trauma, but they may unwittingly eliminate valuable DNA evidence in the process. This study evaluated the effect of 10 maceration methods on gross bone structure and the preservation of DNA in ribs of 12 pigs (Sus scrofa). A scoring system was applied to evaluate the ease of maceration and resulting bone quality while DNA purity was quantified by optical densitometry analysis, followed by polymerase chain reaction (PCR) amplification of three mitochondrial and three nuclear loci. The results demonstrated that while mitochondrial DNA could be amplified for all experiments, cleaning treatments using bleach, hydrogen peroxide, ethylenediaminetetraacetic acid/papain, room temperature water and detergent/sodium carbonate followed by degreasing had low DNA concentrations and failed to generate nuclear PCR products. In general, treatments performed at high temperatures (90 degrees C or above) for short durations performed best. This study shows that traditionally "conservative" maceration techniques are not necessarily the best methods to yield DNA from skeletal tissue.     

Recovery of Environmental Human DNA by Insects*,†

Abstract: We tested the hypotheses that foraging insects can acquire human DNA from the environment and that insect‐delivered human DNA is of sufficient quantity and quality to permit standard forensic analyses. Houseflies, German cockroaches, and camel crickets were exposed to dusty surfaces and then assayed for human mitochondrial and nuclear loci by conventional and qPCR, and multiplex STR amplification. Over two experiments, 100% of insect groups and 94% of dust controls tested positive for human DNA. Of 177 individuals, 33–67% tested positive and 13 yielded quantifiable human DNA (mean = 0.022 ± 0.006 ng; mean dust control = 2.448 ± 0.960 ng); four had at least one positive allele call for one or more locus; eight others showed multiple peaks at some loci. Results imply that application to routine forensic casework is limited given current detection methodology yet demonstrate the potential use of insects as environmental samplers for human DNA.     

Evaluation of Direct PCR Amplification Using Various Swabs and Washing Reagents

DNA profiles were generated via direct amplification from blood and saliva samples deposited on various types of swab substrates. Each of the six non-FTA substrates used in this research was punched with a Harris 1.2 mm puncher. After 0.1 μL of blood or 0.5 μL saliva, samples were deposited on each of these punches, samples were pretreated with one of four buffers and washing reagents. Amplification was performed using direct and nondirect autosomal and Y-STR kits. Autosomal and Y-STR profiles were successfully generated from most of these substrates when pretreated with buffer or washing reagents. Concordant profiles were obtained within and between the six substrates, the six amplification kits, and all four reagents. The direct amplification of substrates which do not contain lysing agent would be beneficial to the forensic community as the procedure can be used on evidence samples commonly found at crime scenes.     

Development of a Tetraplex PCR Assay for CYP2D6 Genotyping in Degraded DNA Samples

CYP2D6 polymorphism analysis is gaining increasing interest in forensic pharmacogenetics. Nevertheless, DNA recovered from forensic samples could be of poor quality and not suitable for long polymerase chain reaction required to type CYP2D6 gene prior to SNaPshot minisequencing analysis performed to define alleles with different enzymatic activity. We developed and validated following the guidelines of the Scientific Working Group on DNA Analysis Methods a tetraplex PCR yielding four amplicons of 597, 803, 1142, and 1659 bp encompassing the entire CYP2D6 gene to analyze eleven SNP positions by SNaPshot minisequencing. Concordance, sensitivity, and specificity were assessed. The method, applied to thirty‐two forensic samples failed to amplify with long PCR, allowed the amplification of CYP2D6 gene in 62.5% of degraded samples. The new tetraplex PCR appears a suitable method for CYP2D6 analysis in forensic pharmacogenetics.