Isolation of DNA from saliva of betel quid chewers using treated cards

Betel quid (BQ) chewing, a common tradition in tropical areas, often poses a problem during collection and DNA analysis of buccal samples from many indigenous communities for population genetic studies and in forensic analysis of chewed BQ residues. This study evaluated the use of FTA card, a chemically treated filter paper, in collecting buccal samples from long-term BQ users and subsequent PCR-based analysis using nine STR markers. A low overall success rate of amplification was observed in the samples extracted using a standard organic extraction procedure (7%) as compared with those prepared using the FTA card (89%). The presence of inhibitors in liquid DNA samples was verified when control DNA failed to amplify in the presence of an equal volume of liquid BQ samples. The use of the FTA card is more practical during field sampling than handling tubes containing buccal swabs.     

烧骨DNA检验技术的研究

目的解决陈旧性骨骼和烧骨DNA检验难题。方法研究建立了CTAB法裂解提取DNA,再用磁珠纯化得到的DNA提取液进行STR复合扩增检验。结果实验结果及实际检案显示研究所建立的骨DNA提取方法能较好地去除DNA扩增抑制物,得到高质量的DNA模板。结论本研究所建立的烧骨DNA检验方法其识别率为10×10-12,达到个人同一认定的目的,在解决实际工作中杀人焚尸案、火灾、爆炸等恶性案件和事故中有重要的作用。     

A simple and efficient method for extracting DNA from old and burned bone

It has been a challenge to extract DNA from bones previously soaked in water, burned, or buried for a long time, due to the reduced quality and quantity of DNA in the bone samples. The dramatic degradation of the DNA and the presence of PCR inhibitors in the collagen significantly complicate the process of DNA identification in dated and charred bones. In this article, we present a novel strategy to obtain DNA from bones based on the use of cetyltrimethylammonium bromide (CTAB) lysis buffer and isoamyl alcohol-chloroform extraction with subsequent DNA purification using the DNA IQ System, or alternatively the QIAquick system. When applied to bones soaked, burned or buried for up to nine years, this method increases the purity and yield of DNA with respect to the traditional phenol-chloroform method and significantly improves multiplex STR genotyping using fluorescence-based methods. The results of this research will assist forensic scientists in the identification of DNA from victims whose bodies underwent significant trauma or burning, precluding the utilization of traditional forensic DNA identification techniques.     

Extraction of high quality DNA from biological materials and calcified tissues

Calcified tissues, such as bone and tooth, and some other sample types, such as those containing adhesive, present a challenge to standard extraction protocols. We have developed a lysis reagent, BTA™ lysis buffer, which is designed for use with PrepFiler™ Kit reagents. The BTA™ lysis buffer disrupts calcified tissue matrices and achieves effective extraction of DNA from pulverized bone and tooth samples. In addition, the BTA™ lysis buffer mildly but efficiently extracts DNA from challenging substrates like tape, chewing gum, and cigarette butts and, as with bone and tooth, DNA from these lysates is purified using established PrepFiler™ reagent extraction protocols.We successfully extracted DNA from powdered human bone samples, chewed gum and smoked cigarettes using BTA™ lysis buffer. Extraction yields for bone, gum and cigarette samples tested were consistent and reproducible. This extraction method efficiently removed potential PCR inhibitors from all samples tested, and C_T values for the internal PCR control of Quantifiler^® Human DNA Quantification Kit were consistent and within the normal range. The DNA extracted from these samples also provided conclusive profiles that were free of PCR artifacts when amplified using the AmpF?STR^® Identifiler^® PCR Amplification Kit. The protocol is easily adapted for automation.     

陈旧性骨骼DNA提取技术的研究

对陈旧性骨骼建立一个高回收率并能除去 PCR 反应抑制物的提取 DNA 的方法。采用 CTAB 法提取 DNA。结果显示,该方法不但能有效去除 PCR 抑制物,而且对水泡、火烧、土埋以及10年左右的骨骼所提取的 DNA 均能成功地进行荧光标记 STR 多基因座扩增检验。实验表明,该方法稳定,重复性好,适合陈旧骨骼标本的 DNA 提取。     

A dedicated automated system for extraction, quantification and STR amplification of forensic evidence samples

Forensic DNA analysis is a multi-step process involving extraction of DNA, quantification of human DNA in the extract, amplification using multiplex STR systems, separation of products, and data analysis. The backlog of forensic casework is increasing worldwide. Automation is one significant way to alleviate the bottleneck of sample processing in forensic labs. The HID EVOlution™ Combination System described here is a robust, reliable sample processing platform, easily adapted to forensic laboratory workflows. Using a variety of forensic sample types including: blood stained FTA paper, cotton fabric and denim, dried blood spiked with known PCR inhibitors, saliva on cotton swabs, and semen stains, we found that yields of human DNA and STR profiles obtained with AmpFlSTR^® Idenitfiler^® kits were complete, highly reproducible, and equivalent to results obtained using the manual PrepFiler™ reagent extraction method. Automated operation was clean, and no cross-contamination was detected between extraction blanks and interspersed high DNA content samples.     

运用酚-氯仿法结合磁珠法提取蝇蛆体内人类DNA

目的建立运用酚-氯仿法结合磁珠法从蝇蛆嗉囊内提取人类DNA的方法,从而提高STR分型检验的灵敏度。方法采用酚-氯仿法对蝇蛆嗉囊内容物中的人类DNA进行提取,提取产物经磁珠法纯化浓缩后用QuantifilerTM人类DNA定量试剂盒在7500型实时荧光定量PCR仪上进行PCR定量,再用AmpF■STR IndentifilerTM试剂盒在3130XL-Avant遗传分析仪上对这些DNA样本进行STR分型。结果本研究建立的方法可增加模板DNA浓度约为单独使用酚-氯仿法的2倍。用此方法提取到的DNA浓度[（0.218±0.041）ng/μL]可获得全部16个STR分型结果。结论酚-氯仿法结合磁珠法可以有效地提高提取到的人类DNA样本STR分型检验的灵敏度,对于从事法医昆虫学方面研究的工作者有较好的实用价值。     

Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods

Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the software methods.     

A more efficient extraction method of human bone resulting in improved DNA profiling

Eight human bone samples, from a forensic case, were extracted in parallel using our standard protocol with and without PTB in the buffer. Both methods were sometimes inadequate for (complete) STR profiling, while the presence of PTB even decreases the DNA yield.The complete decalcification of the bone extraction residues in an EDTA-solution with SDS recovered sufficient amounts of DNA, which resulted in complete STR profiling for all samples. Complete decalcification without SDS yielded even higher amounts of DNA and also complete STR profiling for all samples.Similar results were obtained for the DNA extraction from a human tooth.     

A simple automated instrument for DNA extraction in forensic casework

The Qiagen BioRobot EZ1 is a small, rapid, and reliable automated DNA extraction instrument capable of extracting DNA from up to six samples in as few as 20 min using magnetic bead technology. The San Diego Police Department Crime Laboratory has validated the BioRobot EZ1 for the DNA extraction of evidence and reference samples in forensic casework. The BioRobot EZ1 was evaluated for use on a variety of different evidence sample types including blood, saliva, and semen evidence. The performance of the BioRobot EZ1 with regard to DNA recovery and potential cross-contamination was also assessed. DNA yields obtained with the BioRobot EZ1 were comparable to those from organic extraction. The BioRobot EZ1 was effective at removing PCR inhibitors, which often co-purify with DNA in organic extractions. The incorporation of the BioRobot EZ1 into forensic casework has streamlined the DNA analysis process by reducing the need for labor-intensive phenol-chloroform extractions.     

Simple and highly effective DNA extraction methods from old skeletal remains using silica columns

The recovery of DNA data from old skeletal remains is often difficult due to degraded and very low yield of extracted DNA and the presence of PCR inhibitors. Herein, we compared several silica-based DNA extraction methods from artificially degraded DNA, DNA with PCR inhibitors and DNA from old skeletal remains using quantitative real-time PCR. We present a modified large-scale silica-based extraction combined with complete demineralization, that enables maximum DNA recovery and efficient elimination of PCR inhibitors. This is performed with high concentration of EDTA solution for demineralization of bone powder followed by QIAamp^® spin columns and buffers from the QIAquick^® PCR purification kit. We have successfully used this modified technique to perform STR analysis for 55-year-old skeletal remains. The results of this study will contribute to solve the forensic cases dealing with skeletal remains.     

Use of Embryos Extracted from Individual Cannabis sativa Seeds for Genetic Studies and Forensic Applications

Legal limits on the psychoactive tetrahydrocannabinol (THC) content in Cannabis sativa plants have complicated genetic and forensic studies in this species. However, Cannabis seeds present very low THC levels. We developed a method for embryo extraction from seeds and an improved protocol for DNA extraction and tested this method in four hemp and six marijuana varieties. This embryo extraction method enabled the recovery of diploid embryos from individual seeds. An improved DNA extraction protocol (CTAB3) was used to obtain DNA from individual embryos at a concentration and quality similar to DNA extracted from leaves. DNA extracted from embryos was used for SSR molecular characterization in individuals from the 10 varieties. A unique molecular profile for each individual was obtained, and a clear differentiation between hemp and marijuana varieties was observed. The combined embryo extraction–DNA extraction methodology and the new highly polymorphic SSR markers facilitate genetic and forensic studies in Cannabis.     

AutoMate Express™ forensic DNA extraction system for the extraction of genomic DNA from biological samples

Abstract: The AutoMate Express? Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler? lysis buffer or PrepFiler BTA? lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep? column, the lysate in the sample tubes were loaded onto AutoMate Express? instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework‐type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day‐to‐day operation.     

Genotyping of DNA samples under adverse conditions of Low Copy Number—LCN (formolisados tissue samples and embedded in paraffin)

This paper aims to describe and evaluate a protocol for extraction of DNA (deoxyribonucleic acid) in formalinized tissues and embedded in paraffin for forensics genetic analysis. In outline the method is the removal of paraffin with an organic solvent in 0.3–0.5 mg of the sample of the tissue under study, followed by removal of formaldehyde, rehydration and soon after the extraction of genomic DNA. The extraction is achieved through the stages of cellular lysis, enzymatic digestion of proteins and DNA precipitation in ethanol medium. With the research we can conclude that even when the DNA is present in small quantities in conditions of extreme difficulties in its extraction, as formalinized tissues and embedded in paraffin, the technique of optimizing the extraction of DNA used both to organic extraction as Chelex, for use in the polymerase chain reaction (PCR), and possible the investigation of different samples of human tissue, biological samples, or was obtained under the conditions tested, a DNA with good quality and concentration. The samples were amplified for the mini-STRs loci using the product marketed in multilocus, using a methodology recommended by the supplier and validated for analysis of forensic DNA. Commercial kit was used MiniFiler from Applied Biosystems. The DNA fragments amplified by PCR showed that the extracted DNA had good amplification.     

差异提取试剂盒在混合斑DNA提取中的应用

目的评估差异提取试剂盒对混合斑样本中的精子和上皮细胞DNA分离提取的有效性。方法采用差异提取试剂盒,选择性裂解精细胞和上皮细胞,结合磁珠法分别对人为控制条件下制备的模拟混合样本和案件中的混合斑检材进行精细胞DNA和上皮细胞DNA的分离提取。对所提取的DNA进行定量分析和STR分型。结果该试剂盒能从精子和上皮细胞不同比例的混合斑中提取出高纯度的精细胞和上皮细胞DNA。结论该差异提取试剂盒适用于性侵害案件中混合斑检材的DNA提取。     

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.     

Automated DNA extraction using the QIAsymphony platform: Estimation of DNA recovery from simulated forensic stains

The aim of this study was to evaluate the forensic protocol recently developed by Qiagen for the QIAsymphony automated DNA extraction platform. Samples containing low amounts of DNA were specifically considered, since they represent the majority of samples processed in our laboratory. The analysis of simulated blood and saliva traces showed that the highest DNA yields were obtained with the maximal elution volume available for the forensic protocol, that is 200 μl. Resulting DNA extracts were too diluted for successful DNA profiling and required a concentration. This additional step is time consuming and potentially increases inversion and contamination risks. The 200 μl DNA extracts were concentrated to 25 μl, and the DNA recovery estimated with real-time PCR as well as with the percentage of SGM Plus alleles detected. Results using our manual protocol, based on the QIAamp DNA mini kit, and the automated protocol were comparable. Further tests will be conducted to determine more precisely DNA recovery, contamination risk and PCR inhibitors removal, once a definitive procedure, allowing the concentration of DNA extracts from low yield samples, will be available for the QIAsymphony.     

A method for preparation of DNA suitable for medico-genetic studies from heparinized blood samples

Heparin traditionally used as an anticoagulant for blood preparations is one of the most powerful inhibitors of the polymerase chain reaction. The molecular genetic analysis for the purpose of forensic medical expertise encounters difficulties when DNA preparations obtained from heparinized samples have to be used. In our practical work, we were faced with the necessity to use heparin-treated blood samples. It turned out impossible to eliminated heparin from DNA isolated from these samples by the known methods. In order to obviate this difficulty, we have developed a special method for obtaining DNA from heparinized blood preparations suitable for the purpose of forensic medical expertise. The new technique includes the stages of preliminary extraction and purification of the blood cell fraction.     

3种DNA提取法在污染严重混合斑分型中的应用比较

目的比较Chelex-100法、酚/氯仿法和二氧化硅膜法3种DNA提取法在污染严重混合斑分型中的应用效果。方法从日常案例中收集污染严重的混合斑25份,差异消化法分离精子后同时用Chelex-100法、酚/氯仿法和二氧化硅膜技术3种方法提取DNA,采用PCR-STR技术对D19S253、FGA和CSF1PO 3个基因座进行分型,Gel-Pro软件处理电泳图谱,SPSS软件分析比较不同方法之间的差异。结果采用Chelex-100法提取DNA,25份检材分型结果均未成功;采用酚/氯仿法,25份检材中10份分型成功,3份检材FGA和CSF1PO基因座可分型,4份检材CSF1PO基因座可分型;采二氧化硅膜纯化法,25份检材均成功分型;酚/氯仿法和二氧化硅膜法两种方法比较,结果存在显著性差异(P<0.05)。结论二氧化硅膜纯化技术可以有效去除PCR抑制物,提取的DNA扩增效果明显优于Chelex-100法和酚/氯仿法,具有较高的应用价值。     

Adaptation and evaluation of the PrepFiler™ DNA extraction technology in an automated forensic DNA analysis process with emphasis on DNA yield, inhibitor removal and contamination security

Within the initial step of the forensic DNA analysis process, the DNA extraction efficiency and especially the removal of potential PCR inhibitors is crucial for subsequent steps, e.g. quantification by real-time PCR and amplification of short tandem repeats (STRs). The protocol of the PrepFiler™ Forensic DNA Extraction Kit was optimized for the application on a Tecan liquid handling workstation Freedom EVO^® 150. This modified application of the PrepFiler™ technology was compared with respect to DNA yield, sensitivity and the ability to remove potential PCR inhibitors to an established routine method working on the same liquid handling workstation based on ChargeSwitch^® Technology (CST) from Invitrogen.