氨基比林血痕预试验处理血痕后样本的DNA定量

目的：探讨氨基比林血痕预试验处理血痕后样本DNA含量的变化及对STR分型检测的影响。方法10名健康无关个体EDTA抗凝血液制成滤纸血痕,氨基比林血痕预试验检测,按试验后血样干燥保存时间分30 min、1 h、3 h、6 h、12 h、24 h共6个实验组,并采用磁珠法、QIAcube DNA纯化法、Chelex-100法三种方法提取样本DNA,应用荧光定量PCR检测样本DNA含量,PCR-STR荧光技术进行STR分型。结果提取方法相同时,氨基比林血痕预试验后血样随干燥保存时间的延长,样本DNA含量呈逐渐降低的趋势。保存时间相同时,不同DNA提取方法间,样本DNA含量差异也有统计学意义。90.56%样本均可获得16个STR基因座明确分型。结论氨基比林血痕预试验对血痕样本DNA有损伤,24 h内多可获有效STR分型。磁珠法提取样本DNA进行STR分型,效果最好。     

215例枪支上接触DNA检材的检验分析

目的分析215例枪支上接触DNA提取、送检及检验结果,探讨枪支上接触DNA检出情况及可能影响检验结果的影响因素。方法收集自2013年以来受理的215例涉案枪支上接触DNA检材,按照提取部位、检出率、送检时间、检验方法进行分类并对数据进行统计分析。结果215例接触DNA成功检出35例,检出率为16.28%;枪支上不同部位接触检材的检出率无明显差异;硅膜法与改良硅珠法的检出率无明显差异;送检时间早的检材检出率高于送检时间晚的检材并具有统计学意义。结论枪支上接触DNA的检出率与提取部位、送检时间、检验方法等因素有关,日常类似检材应合理提取、及时送检并采取正确检验方法。     

Elimination of Bioweapons Agents from Forensic Samples During Extraction of Human DNA

Collection of DNA for genetic profiling is a powerful means for the identification of individuals responsible for crimes and terrorist acts. Biologic hazards, such as bacteria, endospores, toxins, and viruses, could contaminate sites of terrorist activities and thus could be present in samples collected for profiling. The fate of these hazards during DNA isolation has not been thoroughly examined. Our goals were to determine whether the DNA extraction process used by the Royal Canadian Mounted Police eliminates or neutralizes these agents and if not, to establish methods that render samples safe without compromising the human DNA. Our results show that bacteria, viruses, and toxins were reduced to undetectable levels during DNA extraction, but endospores remained viable. Filtration of samples after DNA isolation eliminated viable spores from the samples but left DNA intact. We also demonstrated that contamination of samples with some bacteria, endospores, and toxins for longer than 1 h compromised the ability to complete genetic profiling.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

A Powder-free DNA Extraction Workflow for Skeletal Samples

The processing of skeletal material poses several challenges for forensic laboratories. Current methods can be laborious, time-consuming, require dedicated equipment, and are vulnerable to contamination. In this study, various sample mass (1 × 50 mg, 3 × 50 mg, and 1 × 150 mg chip(s)) and incubation times (2, 4, and 16 h) were tested using the PrepFiler^® BTA™ Forensic DNA Extraction Kit to digest whole bone chips in lieu of powdering. The most effective method was then applied to bones and tooth fragments collected from contemporary human cadavers exposed to various environmental conditions using an automated platform. Over a third of the samples tested generated full DNA profiles without having to powder the bone/tooth fragment or further alter the manufacturer's protocol. However, for most samples resulting in incomplete STR profiles due to low amounts of DNA, slightly better results were achieved with powdered tissue. Overall, this work demonstrates the potential use of a faster, nonpowdering DNA extraction method for processing skeletal samples as an effective first-pass screening tool.     

实时DNA定量技术的应用研究

通过应用TaqMan探针法和SYBR Green荧光染料法对常见的法医生物学检材进行DNA定量,对实时荧光定量PCR技术用于法庭科学样品定量检测的适用性进行研究。实验结果表明,该技术在DNA定量及抑制因素评估等方面具有重要的法医学应用价值。     

The Influence of Swabbing Solutions on DNA Recovery from Touch Samples,

There has been minimal research into how to best obtain DNA from touch samples. Many forensic laboratories simply moisten a swab with water and use it for collecting cells/DNA from evidentiary samples. However, this and other methods have not been objectively studied in order to maximize DNA yields. In this study, fingerprints were collected using swabs moistened with water or laboratory or commercially available detergents, including sodium dodecyl sulfate (SDS), Triton X‐100, Tween 20, Formula 409^®, and Simple Green^®. Prints were swabbed, DNA isolated using an organic extraction, yields quantified, and relative yields compared. In all cases, the detergent‐based swabbing solutions outperformed water, with SDS and Triton X‐100 producing significant increases in yield. Short tandem repeat profiles were consistent with the individuals that placed them. Subsequent analysis of SDS concentrations for collecting touch DNA demonstrated an increase in DNA yield with increasing SDS concentration, with an optimal concentration of approximately 2%.     

Evaluation of Skin Surface as an Alternative Source of Reference DNA Samples: A Pilot Study

An acceptable area for collecting DNA reference sample is a part of the forensic DNA analysis development. The aim of this study was to evaluate skin surface cells (SSC) as an alternate source of reference DNA sample. From each volunteer (n = 10), six samples from skin surface areas (forearm and fingertips) and two traditional samples (blood and buccal cells) were collected. Genomic DNA was extracted and quantified then genotyped using standard techniques. The highest DNA concentration of SSC samples was collected using the tape/forearm method of collection (2.1 ng/μL). Cotton swabs moistened with ethanol yielded higher quantities of DNA than swabs moistened with salicylic acid, and it gave the highest percentage of full STR profiles (97%). This study supports the use of SSC as a noninvasive sampling technique and as a extremely useful source of DNA reference samples among certain cultures where the use of buccal swabs can be considered socially unacceptable.     

PE‐Swab Direct STR Amplification of Forensic Touch DNA Samples

The PE‐Swab direct STR amplification workflow was developed to process low‐level “touch DNA” samples. In this workflow, a forensic sample is first collected on a 4‐mm PE‐Swab (a novel sample collection device); two 2‐mm punches containing collected samples are then generated from the PE‐Swab and directly amplified for STR typing. Compared to the conventional STR workflow, which involves DNA extraction, purification, and elution volume reduction, the PE‐Swab direct STR amplification workflow does not require sample preparation and takes <60 sec before a touch sample is ready for STR amplification. Because there is no DNA loss due to sample preparation, the PE‐Swab workflow is more sensitive than the conventional STR workflow. The average peak height per sample obtained by the PE‐swab workflow is 3 times higher than that from the conventional workflow with both low‐level single source and two‐contributor mixture samples tested in this study.     

微量生物物证提取套装在现场微量血痕DNA检验中的应用

目的探讨微量生物物证提取套装应用于提取现场微量血痕DNA的检验效果。方法将静脉血制成地面血痕。分别应用微量生物物证提取套装法和普通法提取血痕。分别于恒温摇床放置2、24、48、72、96h(每组50份)进行血痕DNA检验,对比各组检验结果。结果恒温摇床上分别放置24、48、72、96h后,微量生物物证提取套装法的DNA检出率(分别为100.00%、100.00%、100.00%、96.00%)均高于普通法(分别为62.00%、26.00%、10.00%、0),差异均具有统计学意义(P<0.05)。恒温摇床上放置2h,两种方法的DNA检出率差异无统计学意义(P>0.05)。结论微量生物物证提取套装可有效提升放置时间较久的现场微量血痕的检出率和检出时限。     

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.     

Age Estimation Based on DNA Methylation Using Blood Samples From Deceased Individuals

Age estimation using DNA methylation levels has been widely investigated in recent years because of its potential application in forensic genetics. The main aim of this study was to develop an age predictor model (APM) for blood samples of deceased individuals based in five age-correlated genes. Fifty-one samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for DNA methylation evaluation in genes ELOVL2, FHL2, EDARADD, PDE4C, and C1orf132. Linear regression was used to analyze relationships between methylation levels and age. The model using the highest age-correlated CpG from each locus revealed a correlation coefficient of 0.888, explaining 76.3% of age variation, with a mean absolute deviation from the chronological age (MAD) of 6.08 years. The model was validated in an independent test set of 19 samples producing a MAD of 8.84 years. The developed APM seems to be informative and could have potential application in forensic analysis.     

Use of a Spray Device to Locate Touch DNA on Casework Samples

The use of a fluorescent dye to visualize cellular material on surfaces offers a targeted sampling approach for locating touch DNA on casework items. However, the current application of such dye is not feasible for examination of relatively large items. As a result, development of an efficient dye application system is required to translate this approach into practice. Here, the spray pattern (area covered, intensity, and evenness) of 15 different commercial spray devices was examined visually using food coloring. From this, five devices were selected to apply Diamond Nucleic Acid Dye (DD) to three substrates (glass slide, plastic sheet, and brown packing tape) seeded with saliva and touch DNA. The cellular material was visualized using the Dino-lite Microscope and Polilight. The inhibitory effects of DD afforded by each spray device were examined using Identifiler Plus^® DNA profiling kit and a DNA input of 800 pg. The two most promising devices were further tested on a range of mock casework items seeded with touch DNA. The results presented demonstrate the feasibility of a spray system to apply DD to large surfaces and subsequently detect cellular material at both micro and macroscale. Specifically, the data suggest that a pressurized continuous-spray system is favorable and that droplet size influences the intensity of fluorescence and surface coverage. Furthermore, this study indicates that full STR profiles can be obtained following spraying with DD solution, even with excessive application, which is essential for the widespread use of these devices in casework.     

Applicability of the ParaDNA® Screening System to Seminal Samples

Seminal fluid represents a common biological material recovered from sexual assault crime scenes. Such samples can be prescreened using different techniques to determine cell type and relative amount before submitting for full STR profiling. The ParaDNA^® Screening System is a novel forensic test which identifies the presence of DNA through amplification and detection of two common STR loci (D16S539 and TH01) and the Amelogenin marker. The detection of the Y allele in samples could provide a useful tool in the triage and submission of sexual assault samples by enforcement authorities. Male template material was detected on a range of common sexual assault evidence items including cotton pillow cases, condoms, swab heads and glass surfaces and shows a detection limit of 1 in 1000 dilution of neat semen. These data indicate this technology has the potential to be a useful tool for the detection of male donor DNA in sexual assault casework.     

生物检材DNA碱性裂解提取法的优化与改良

目的通过对DNA碱性裂解提取法进行优化和改进,建立一种操作更简便、检验快速、结果更可靠的生物检材DNA提取方法。方法以抗凝血作为检验样本,通过改变提取试剂的浓度、pH值、保存时间及孵化样本的温度、时间等条件,观测对检验结果峰值的影响,以确定提取试剂最佳条件,DNA提取物最佳保存条件。通过用改良后的碱性裂解法及Chelex100法同时提取不同量的抗凝血样本、各类法医生物检材,用不同厂家的DNA试剂盒扩增,对碱性裂解法的灵敏度、适应性和适用性进行评估。结果改良后的碱性裂解法只需将生物检材用NaOH（0.25mol/L）99℃孵化8min,振荡后加入TrisHCl（0.05mol/L,pH=6.5）中和液,离心后直接扩增检测。DNA提取物于-20℃冰箱可长期保存。对于毛发及精斑类检材优于Chelex100法。DNA提取物适用于现有各种DNA试剂盒扩增检测,其灵敏度与Chelex100法相似。结论改良后的碱性裂解法,操作简便、检验快速、结果可靠,可适合于法庭科学的检案与建库。     

Resistance of degraded hair shafts to contaminant DNA

We have investigated the susceptibility of degraded human hair shaft samples to contamination by exogenous sources of DNA, including blood, saliva, skin cells, and purified DNA. The results indicate that on the whole hair shafts are either largely resistant to penetration by contaminant DNA, or extremely easy to successfully decontaminate. This pertains to samples that are both morphologically and biochemically degraded. We suggest that this resistance to the incorporation of contaminant DNA relates to the hydrophobic and impermeable nature of the keratin structures forming the hair shaft. Therefore, hair samples represent an important and underestimated source of DNA in both forensic and ancient DNA studies.     

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.     

Developmental Validation of the ANDE 6C System for Rapid DNA Analysis of Forensic Casework and DVI Samples

A developmental validation was performed to demonstrate reliability, reproducibility, and robustness of the ANDE Rapid DNA Identification System for processing of crime scene and disaster victim identification (DVI) samples. A total of 1705 samples were evaluated, including blood, oral epithelial samples from drinking containers, samples on FTA and untreated paper, semen, bone, and soft tissues. This study was conducted to address the FBI’s Quality Assurance Standards on developmental validation and to accumulate data from a sufficient number of unique donors and sample types to meet NDIS submission requirements for acceptance of the ANDE Expert System for casework use. To date, no Expert System has been approved for such samples, but the results of this study demonstrated that the automated Expert System performs similarly to conventional laboratory data analysis. Furthermore, Rapid DNA analysis demonstrated accuracy, precision, resolution, concordance, and reproducibility that were comparable to conventional processing along with appropriate species specificity, limit of detection, performance in the presence of inhibitors. No lane-to-lane or run-to-run contamination was observed, and the system correctly identified the presence of mixtures. Taken together, the ANDE instrument, I-Chip consumable, FlexPlex chemistry (a 27-locus STR assay compatible with all widely used global loci, including the CODIS core 20 loci), and automated Expert System successfully processed and interpreted more than 1200 unique samples with over 99.99% concordant CODIS alleles. This extensive developmental validation data provides support for broad use of the system by agencies and accredited forensic laboratories in single-source suspect-evidence comparisons, local database searches, and DVI.     

Knowledge on DNA Success Rates to Optimize the DNA Analysis Process: From Crime Scene to Laboratory

DNA analysis has become an essential intelligence tool in the criminal justice system for the identification of possible offenders. However, it appears that about half of the processed DNA samples contains too little DNA for analysis. This study looks at DNA success rates within 28 different categories of trace exhibits and relates the DNA concentration to the characteristics of the DNA profile. Data from 2260 analyzed crime samples show that cigarettes, bloodstains, and headwear have relatively high success rates. Cartridge cases, crowbars, and tie‐wraps are on the other end of the spectrum. These objective data can assist forensics in their selection process.The DNA success probability shows a positive relation with the DNA concentration. This finding enables the laboratory to set an evidence‐based threshold value in the DNA analysis process. For instance, 958 DNA extracts had a concentration value of 6 pg/μL or less. Only 46 of the 958 low‐level extracts provided meaningful DNA profiling data.     

Trace DNA success rates relating to volume crime offences

In this study, 252 trace DNA samples (from handled surfaces) from 201 burglary, robbery and drugs cases were compiled to assess success rates and to interpret the value of trace DNA evidence in volume crime investigations. The average amount of DNA recovered from the trace DNA samples collected was 1.7 ng. Full or major (12 or more alleles) profiles were recovered from 14% of samples. Samples from firearms and burglary points of entry were the least successful. Mixtures were recovered from 21% of samples, presenting a case for the collection of more elimination profiles to enable more samples to be used for database purposes. The research highlighted the difficulties in collecting data relating to the success rates of samples. Computerised automation of this process would be extremely beneficial in the assistance of policy development, method application, training, and investigative usefulness.