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.     

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.     

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.     

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.     

Multicolor Real-Time PCR Genotyping of ABO System Using Displacing Probes

Abstract:  Rapid and informative ABO genotyping has become increasingly popular in forensic use. We developed a multiplex real-time polymerase chain reaction (PCR) approach to genotype ABO major groups and subgroups. Seven differently fluorophor-labeled displacing probes for O¹(261delG), A(261G), A(796C/803C), B(796A/803C), O² (802G>A), A² (1059delC), and A² (1009A>G) were combined in one or two PCRs to determine either ABO major groups or subgroups. The method correctly detected 13 reference DNA samples. A blind test of 237 samples resulted in complete agreement with their phenotypes, and 110 of these 237 samples as well as with PCR-SSP method. The whole analysis could be finished in less than 100 min at substantially low material cost and the template DNA ranging from 0.16 to 500 ng per reaction could be quantitatively detected. Despite the limited informativeness of ABO genotyping, the developed methods could find application in rapid and inexpensive screening of forensic settings.     

Recovery and STR amplification of DNA from RFLP membranes

Restriction fragment length polymorphism (RFLP) techniques were utilized in the forensic DNA community until the mid 1990s when less labor-intensive polymerase chain reaction short tandem repeat (PCR STR) techniques became available. During the transition from RFLP technology to PCR-based STR platforms, a method for comparing RFLP profiles to STR profiles was not developed. While the preferred approach for applying new technology to old cases would be to analyze the original biological stain, this is not always possible. For unsolved cases that previously underwent RFLP analysis, the only DNA remaining may be restriction cut and bound to nylon membranes. These studies investigate several methods for obtaining STR profiles from membrane bound DNA, including removal of bound DNA with bases, acids, detergents, various chemicals, and conventional cell extraction solutions. Direct multiplex STR amplification of template in the membrane-bound state was also explored. A partial STR profile was obtained from DNA that was recovered from an archived membrane using conventional extraction buffer components, indicating promise for recovering useful STR information from RFLP membranes that have been maintained in long-term frozen storage.     

Comparative Analysis of the Effects of Heat on the PCR‐Amplification of Various Sized DNA Fragments Extracted from Sus Scrofa Molars*

Abstract: This study examined the effects of heat on the amplification of DNA from the dental pulp of Sus scrofa molars and investigated the protection afforded to the pulp tissue by the dental enamel, alveolar process, and soft tissue of the head. Segments of defleshed maxilla and mandible encasing the first molar (n = 60) were subject to a range of temperatures for 15 min. Dental pulps were retrieved. Amplifications using three‐primer and four‐primer multiplexes showed no degradation of the largest fragment following exposure to 450°C. Amplifications in the three‐primer multiplex (283 bp) were successful following exposure to 525°C in maxillary samples only. This study revealed the enamel density of maxillary molars to be greater than mandibular molars in Sus scrofa. Following incineration of intact heads for 15 min (n = 10) and 1 h (n = 4) at an average temperature of 625°C, amplifications of the largest fragment (450 bp) were successful from both maxillary and mandibular teeth.     

InDel_typer30:用于中国5个主要民族DNA鉴定的多重PCR系统

目的采用插入/缺失(Insertion/Deletion,InDel)多态性遗传标记,建立一种可用于中国汉族、回族、维吾尔族、蒙古族、藏族5个主要民族法医DNA鉴定的多重PCR系统。方法采用人类基因组浏览器和dbSNP数据库筛选具有高度遗传多态性的人类常染色体InDel标记,采用Primer 3软件设计多重PCR引物,通过复合荧光标记系统建立多重PCR扩增体系,采用该体系对汉、回、维、蒙、藏5个民族进行多态性调查。结果成功建立了一个包含30个InDel位点和Amelogenin性别鉴定位点的复合荧光多重PCR扩增体系,命名为InDel_typer30。多态性调查显示这30个InDel位点在上述5个主要民族中均呈高度遗传多态性,平均期望杂合度分别为:0.464、0.460、0.453、0.466和0.469,平均个人识别率分别为:0.595、0.585、0.586、0.589和0.595。该系统在5个民族中的累积个人识别率(CDP)均达到0.999999999996以上。结论 InDel_typer30是一种适用于中国汉、回、维、蒙、藏5个主要民族的法医DNA鉴定系统。     

A LDR‐PCR Approach for Multiplex Polymorphisms Genotyping of Severely Degraded DNA with Fragment Sizes <100 bp*

Abstract: Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini‐short tandem repeat‐polymerase chain reaction (PCR) and mini‐sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small‐scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.     

Direct STR amplification from whole blood and blood- or saliva-spotted FTA without DNA purification

The DNA purification step has been thought to be essential for typing of STR DNA. However, this process is time-consuming, and there is a risk of unexpected cross-contamination during purification. We report a new method for direct short tandem repeat (STR) amplification using a newly developed direct PCR buffer, AnyDirect, which can amplify STR loci from whole blood and blood- or saliva-spotted FTA cards without DNA purification. The autosomal and Y chromosomal STR loci were analyzed for whole blood and blood or saliva spots of random individuals, followed by comparison of the results with those of corresponding purified DNA. The results from whole blood and blood spots showed perfect concordance with those from purified DNA without allele or locus drop-out. However, in the case of saliva spots, no amplification or locus drop-out was observed in some of the samples, which offers a topic for further study. Additionally, some commercial hot-start DNA polymerases other than AmpliTaq Gold DNA polymerase were also found to be compatible with this buffer system. Therefore, this direct PCR buffer was demonstrated to be useful for fast forensic DNA analysis or criminal DNA databases for which there is no need to store DNA samples.