Quantitation of human genomic DNA through amplification of the amelogenin locus

An alternate method for quantitation of human genomic DNA is presented. Quantitative template amplification technology (abbreviated "Q-TAT") estimates the quantity of human DNA present in an extract by comparing fluorescence in X and Y amplicons produced from unknowns with fluorescence in a standard curve amplified from known quantities of reference DNA. Q-TAT utilizes PCR and electrophoresis with fluorescent detection/quantitation, precluding the need for new instrumentation, methodology, or quality assurance associated with slot-blot or real-time PCR. In a comparison study incorporating shared samples, Q-TAT was found to be more sensitive than widely used slot-blot methods but somewhat less sensitive than real-time PCR. Among samples containing DNA concentrations ranging from 100 pg/microL to 2-4 ng/microL, Q-TAT produced DNA concentration estimates that agreed reasonably well with either Quantiblot or real-time PCR. Q-TAT was reproducible with a typical coincidence of variation of about 35%. Quantitation of human DNA in this study involved summing fluorescence in X and Y amplicons in unknowns and quantitation standards. However, analyzing fluorescence in X and Y amplicons individually could allow estimates of male and female DNA present in mixtures to be made. Moreover, since X and Y amplicons exhibit sizes of 210 and 216 bp, respectively, the integrity as well as the concentration of the genomic DNA template can be assessed. Q-TAT represents an alternate method useful for the quantitation of human genomic DNA prior to amplification of STR loci used for identity testing purposes. The method uses existing equipment and procedures in conjunction with a well-characterized DNA standard to produce concentration estimates for unknowns that reliably produce STR profiles suitable for analysis.     

Validation of a DNA Quantitation Method on the Biomek® 3000

Abstract: Laboratory automation has the ability to increase the throughput and efficiency of laboratory processes to keep pace with current backlogs and requests for analysis. This paper addresses the specific studies employed to properly evaluate an automated method for DNA quantitation setup using Applied Biosystems Quantifiler? Human DNA Quantification kit on a Biomek^® 3000. The calibration of robotic pipetting as well as comparison with manually performed steps confirmed the accuracy of the automated methods used. Reproducibility studies evaluated differences between robotic and manually prepared human DNA standard curves. Additional studies examined DNA samples of known quantities, extract storage formats, sensitivity, and an assessment of contamination. The Biomek^® 3000 not only demonstrated reproducibility and accuracy that equaled or surpassed the manual method but also revealed a contamination‐free method to replace the multiple pipetting steps required during quantitation setup.     

Additional sequence characterization of NIST SRM 2391c: PCR-Based DNA Profiling Standard

The NIST Standard Reference Material (SRM) 2391c: PCR-Based DNA Profiling Standard was designed for use in the standardization of forensic and paternity quality assurance procedures for fragment-based typing short tandem repeat (STR) alleles generated by the polymerase chain reaction (PCR). Certified genotypes of the 6 components A–F were assigned for 24 autosomal and 17 Y-STR markers plus Amelogenin using concordance testing between commercial kits. Selected Sanger sequencing characterization was performed for the alleles of 11 STR markers when only one PCR primer set was available for fragment-based typing. The goal is to characterize the remaining 30 STR loci in components A–C by Sanger sequencing methods for the STR repeat regions and adjacent flanking regions. Additional characterization of the SRM is intended to support the emerging interest in next-generation sequencing technologies for forensic typing applications. Sanger methods have detected underlying polymorphisms (sequence, insertion-deletion, variation in complex motifs) typically not detected by fragment-based typing. The sequenced regions include the commercial or known PCR binding sites commonly implemented in fragment-based typing.     

Results of the 2003-2004 GEP-ISFG collaborative study on mitochondrial DNA: focus on the mtDNA profile of a mixed semen-saliva stain

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003-2004. Five reference bloodstains from five donors (M1-M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1-M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1-M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.     

Results of the 2018 Rapid DNA Maturity Assessment

Three commercially available integrated rapid DNA instruments were tested as a part of a rapid DNA maturity assessment in July of 2018. The assessment was conducted with sets of blinded single-source reference samples provided to participants for testing on the individual rapid platforms within their laboratories. The data were returned to the National Institute of Standards and Technology (NIST) for review and analysis. Both FBI-defined automated review (Rapid DNA Analysis) and manual review (Modified Rapid DNA Analysis) of the datasets were conducted to assess the success of genotyping the 20 Combined DNA Index System (CODIS) core STR loci and full profiles generated by the instruments. Genotype results from the multiple platforms, participating laboratories, and STR typing chemistries were combined into a single analysis. The Rapid DNA Analysis resulted in a success rate of 80% for full profiles (85% for the 20 CODIS core loci) with automated analysis. Modified Rapid DNA Analysis resulted in a success rate of 90% for both the CODIS 20 core loci and full profiles (all attempted loci per chemistry). An analysis of the peak height ratios demonstrated that 95% of all heterozygous alleles were above 59% heterozygote balance. For base-pair sizing precision, the precision was below the standard 0.5 bp deviation for both the ANDE 6C System and the RapidHIT 200.     

Results of the 2003–2004 GEP-ISFG collaborative study on mitochondrial DNA: Focus on the mtDNA profile of a mixed semen-saliva stain

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003–2004. Five reference bloodstains from five donors (M1–M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1–M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1–M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.     

Development and usage of a NIST standard reference material for real time PCR quantitation of human DNA

National Institute of Standards and Technology SRM 2372 human DNA quantitation standard has been produced to support the need for a human-specific DNA quantitation standard in forensic casework and calibration of new quantitative polymerase chain reaction (qPCR) assays. The conventional DNA concentration has been assigned with one of the U.S. National Reference UV/Visible Spectrophotometers, assuming an absorbance of 1.0 at 260 nm equals 50 ng/μL of double stranded DNA. In addition, an interlaboratory study has been conducted, to verify that the SRM 2372 materials perform well in currently used DNA quantitation assays by the forensic DNA community. Each unit of SRM 2372 consists of three well-characterized DNA extracts. Component A is a single-source human male material derived from blood. Component B is a multiple-source human female material derived from blood. Component C was purchased as a purified unsheared genomic human DNA (Sigma-Aldrich Co., St. Louis, MO) obtained as a lyophilized human genomic extract and has both male and female donors. SRM 2372 is intended to enable the comparison of DNA concentration measurements across time and place. Manufacturers can use SRM 2372 to validate the values assigned to their own reference materials. Individual forensic laboratories can use SRM 2372 to validate DNA quantitation methods and to verify the assigned concentration of in-house or commercial DNA calibration standards.     

烟蒂DNA分型的研究

目的 研究烟蒂中DNA提取及其检验。方法 用Chelex-100法提取170枚烟蒂样本的DNA,进行PCR扩增及STR检验。结果 除1名志愿者提供的21枚烟蒂外层纸未能检出STR基因分型外,其余烟蒂外层纸均得到分型结果。加入少许烟丝的样本未能检出STR分型,与口唇接触的海绵有时可检出基因型,6个月内的烟蒂可检出小片段基因座。结论 烟蒂能进行DNA分型,在法医检案中具有应用价值。     

指甲DNA的STR分型研究

目的 研究尸体所处环境、指甲DNA提取部位及提取方法对STR分型的影响。方法 对案件中不同条件腐败尸体指甲,使用不同部位、不同体系和时间消化,酚-氯仿法提取指甲核DNA,进行PCR扩增及STR分型。结果 提取指甲甲体、甲根部位均可获得STR分型。结论 指甲是法医检案中一类具有实用价值的检材。     

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.     

Qubit?快速荧光定量系统在法医学中的应用

目的 研究Qubit 定量系统在法医学中的应用.方法 使用Quant-iTTM试剂盒检测法医DNA样本并得出样本浓度.结果 Quant-iTTM试剂盒能检测并定量各种生物学检材DNA样本.结论 Quant-iTTM试剂盒能够应用在法医学检验中.     

Resiliency against victimization: Results from the National Longitudinal Study of Adolescent Health

Investigating the causes of why individuals desist from, or are resilient to, delinquency, crime, and other problem behaviors has captured the interests of scholars. Within the context of criminology, much of this research focused on resiliency against offending; that is, understanding how and why some individuals within high-risk environments do not engage in serious criminal offenses. The extant scholarship, however, has not fully explored the effects protective factors might have on fostering resiliency against victimization. Using a sample of respondents drawn from the National Longitudinal Study of Adolescent Health, this study investigated how individual protective factors and the accumulation of protective factors contribute to the explanation of resiliency against victimization. Analysis of the data revealed that commitment to school was the only statistically significant independent predictor of resiliency for at risk-individuals. Additional analyses indicated that a protective factor index measuring the accumulation of protection was significant across multiple measures of resiliency. The policy and theoretical implications of these findings are discussed.     

RNA/DNA co-analysis from blood stains—Results of a second collaborative EDNAP exercise

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.     

组织切片DNA提取的探讨

在医患关系纠纷中,病理组织切片经常被作为至关重要的证据,但对它的检验是目前法医DNA检验的难点。因样本制作固定的时间、保存环境等不同因素的影响,病理组织切片DNA检出率并不高。本文从实际检案角度出发,详细介绍了病理组织切片DNA提取的过程,并分析了其可能影响因素。     

Qubit~快速荧光定量系统在法医学中的应用

目的研究Qubit定量系统在法医学中的应用。方法使用Quant-iTTM试剂盒检测法医DNA样本并得出样本浓度。结果Quant-iTTM试剂盒能检测并定量各种生物学检材DNA样本。结论Quant-iTTM试剂盒能够应用在法医学检验中。     

Uses of the NIST 26plex STR assay for human identity testing

Ongoing work at the U.S. National Institute of Standards and Technology has focused on the characterization of 26 autosomal STR loci for human identity testing. These 26 loci are in addition to the existing 13 U.S. core loci and those found in PowerPlex16 and Identifiler commercial STR typing kits. The amplification of the 26 loci has been optimized for degraded extracts in unique miniplex panels and also for reference samples as a single reaction 26plex assay. A study has been performed comparing genotypes obtained with the 26plex primers to those with miniplex panels for allele drop out and concordance. The forensic utility of the 26plex assay was evaluated for situations where additional loci are beneficial. The utility of this large multiplex was also tested in a case involving DNA extracted from degraded bone samples. The 26plex can serve as a low-cost assay (compared to commercially available kits) useful for both sorting comingled remains and providing additional markers for increased statistical support for samples that require “non-trio” family references for human identification.     

Y-STR analysis on DNA mixture samples—Results of a collaborative project of the ENFSI DNA Working Group

The ENFSI (European Network of Forensic Science Institutes) DNA Working Group undertook a collaborative project on Y-STR typing of DNA mixture samples that were centrally prepared and thoroughly tested prior to the shipment. Four commercial Y-STR typing kits (Y-Filer, Applied Biosystems, Foster City, CA, USA; Argus Y Nonaplex, Biotype, Dresden, Germany; Powerplex Y, Promega, Madison, WI, USA; and DYSplex-3, SERAC, Bad Homburg, Germany) were used for the amplification of the mixture samples. The results of the study showed a striking inter-laboratory difference of kit performance as determined from the peak heights of the obtained Y-STR genotypes. Variation in quantity and quality of the shipped DNA can be excluded as reason for the observed differences because both samples and shipping conditions were found to be reproducible in an earlier study. The results suggest that in some cases a laboratory-specific optimization process is indicated to reach a comparable sensitivity for the analysis of minute amounts of DNA.     

New resources for the forensic genetics community available on the NIST STRBase website

For more than a decade, the U.S. National Institute of Standards and Technology (NIST) has maintained the short tandem repeat DNA Internet database (STRBase), which is located at http://www.cstl.nist.gov/biotech/strbase/. The purpose of STRBase has been and continues to be an attempt to bring together the abundant literature and information in the forensic genetics field in a cohesive fashion to make current and future work easier. New materials are regularly added to expand the valuable information contained on the STRBase website.     

Some mathematical problems in the DNA identification of victims in the 2004 tsunami and similar mass fatalities

DNA is a major and essential identification tool for mass fatality incidents including the hundreds of thousands of victims of the 2004 Indian Ocean tsunami. Mathematical complications characteristic of this sort of mass fatality include prevalence of related victims, the many races represented among the victims, and various identification modalities in tandem with DNA. Four mathematical problems of interest are discussed in this paper. (1) Other quantifiable factors (i.e. geography) can be formally accounted for by including a likelihood ratio that can be thought of as reducing the "effective number of victims." (2) When a victim is found and tentatively identified as V, but then it comes to light that the victim has a relative W who is also missing, confidence in the identity is depressed. To account for the existence of W, increment the effective number of victims by the likelihood ratio supporting W as the identity of the victim. (3) When several apparently related victims are found, their mutual identities should be calculated simultaneously. Compared to one-at-a-time, serial identifications, this is both logical and may lead to much more confidence in the identities. (4) Although there may be many different population groups represented among the missing, it is generally sufficient to consider population statistics for only a few of them in deciding whether to declare an identification.     

Effects of Delinquency on Alcohol use Among Juveniles in Europe: Results from the ISRD-2 Study

The existence of a correlation between the use of alcohol and juvenile delinquency has long been acknowledged. In order to evaluate the strength and the characteristics of this association in various cultural contexts, we analysed data collected as part of the International Self-Report Delinquency Study- 2 (ISRD-2). The sample consisted of of young people (N?=?57,771) of both sexes, aged between 12 and 16 years, in 25 European countries. After estimating the prevalence of alcohol consumption among young people involved in property offences and violent offences, we assessed the degree to which these types of delinquency were associated with the use of alcohol in the 25 countries. In addition, we attempted to ascertain the influence of belonging to various types of deviant groups on alcohol use. To this end, we used a Mokken Scale Analysis. With this method, we constructed a scale of “gangness” and correlated the scores with alcohol use among juveniles. The results yielded by the present study indicate that alcohol use and delinquency are closely related with one another. In particular, we observed that alcohol consumption seems to be strongly influenced by involvement in delinquent activities. The nature and characteristics of these relationships suggest that the associations between alcohol use and delinquency are reciprocal rather than one-directional. Consequently, alcohol use constitutes a risk factor for criminal behaviour. Likewise, involvement in delinquency increase the risk of alcohol consumption and, especially, of alcohol abuse.