Automated processing of forensic casework samples using robotic workstations equipped with nondisposable tips: contamination prevention

An automated process has been developed for the analysis of forensic casework samples using TECAN Genesis RSP 150/8 or Freedom EVO liquid handling workstations equipped exclusively with nondisposable tips. Robot tip cleaning routines have been incorporated strategically within the DNA extraction process as well as at the end of each session. Alternative options were examined for cleaning the tips and different strategies were employed to verify cross-contamination. A 2% sodium hypochlorite wash (1/5th dilution of the 10.8% commercial bleach stock) proved to be the best overall approach for preventing cross-contamination of samples processed using our automated protocol. The bleach wash steps do not adversely impact the short tandem repeat (STR) profiles developed from DNA extracted robotically and allow for major cost savings through the implementation of fixed tips. We have demonstrated that robotic workstations equipped with fixed pipette tips can be used with confidence with properly designed tip washing routines to process casework samples using an adapted magnetic bead extraction protocol.     

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 Concentrated Hydrochloric Acid-based Method for Complete Recovery of DNA from Bone

The successful extraction of DNA from historical or ancient animal bone is important for the analysis of discriminating genetic markers. Methods used currently rely on the digestion of bone with EDTA and proteinase K, followed by purification with phenol/chloroform and silica bed binding. We have developed a simple concentrated hydrochloric acid-based method that precludes the use of phenol/chloroform purification and can lead to a several-fold increase in DNA yield when compared to other commonly used methods. Concentrated hydrochloric acid was shown to dissolve most of the undigested bone and allowed the efficient recovery of DNA fragments <100 bases in length. This method should prove useful for the recovery of DNAs from highly degraded animal bone, such as that found in historical or ancient samples.     

法医物证DNA自动化检验技术体系的研究

目的建立自动化工作站同步提取不同种类涉案法医生物检材DNA的新方法。方法选用TECAN Freedom EVO100．4、75—2型自动化提取、加样工作站,采用磁珠法及Chelex-100法对各类涉案生物检材进行DNA提取、PCR扩增、毛细管电泳检测其STR分型,进行比较测试。在“全国公安机关DNA数据库应用系统”中建立并应用实验室信息管理系统（LIMS）模拟实施规范化DNA检案。结果1552份各类检材,采用工作站-磁珠法提取DNA效果最佳,STR检测成功率为95％,工作站-Chelex法为88％;二者分别与其手工提取法比较,成功率无明显差异。92个样本同期检测,自动化工作站较手工操作DNA检案时间可缩减1．25倍。结论工作站域珠法提取涉案检材DNA,可获得满意的STR分型结果。应用LIMS管控,可有效防控污染,明显提高检案效率及鉴定质量。     

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.     

Optimization of recovery of human DNA from envelope flaps using DNA IQ™ System for STR genotyping

An optimized protocol based on the DNA IQ™ System has been tested for the extraction of DNA from envelope flaps. DNA is extracted directly without the need for opening and swabbing the flaps. The method is repeatable with <10% R.S.D. (relative standard deviation). The results of a systematic study show that it is an equilibrium extraction, and a small sample volume as well as high lysis buffer content in sample contribute to high extraction efficiency. The extracted DNA requires no further purification steps following its extraction with the DNA IQ™ System. Complete but skewed 15-locus short tandem repeat (STR) profiles, which is typical of degraded of DNA, have been generated from the DNA extracted from 6 to 9 years old casework envelope samples.     

磁珠法自动化纯化现场检材DNA方法研究

目的利用TE-MAGS在TECAN工作站上结合磁珠试剂盒,建立自动化工作站批量纯化现场检材DNA的方法,并探讨其在法医物证检案中的应用。方法灵敏度测试:标准品使用0.1ng/μL 9947A,用200μL TES稀释制备DNA总量0.1ng~1ng共10种的标准样品,采用本文方法提取纯化,使用IdentifilerTM试剂盒扩增,用3130XL型测序仪检测,Gene Mapper ID-X分析,分析STR图谱质量;纯化能力测试:在1ng总量的标准样品中加入腐殖酸、血红素,采用本文方法提取纯化、扩增检测,分析STR图谱质量;实际案件应用对比:收集304份现场检材,分别采用本方法和硅珠法进行提取纯化,经扩增检测,统计对比两种提取纯化方法 STR分型成功率。结果灵敏度测试:0.1ng~0.2ng总量标准样品提取的DNA模板,扩增后可检测到部分基因座STR图谱,0.3ng~1ng总量标准样品提取的DNA模板,扩增后可以得到完整的STR图谱;纯化能力测试:对混合有一定浓度的腐殖酸、血红素的标准样品的提取产物检测图谱未见明显抑制;实际案件应用对比测试:304份现场检材工作站磁珠法检出成功率(50%)高于硅珠法(40.8%)。结论本文所建立的方法缓冲范围较大,回收率高,纯化能力强,提取产物STR分型成功率高,适合现场检材批量化DNA检验。     

Application of BioRobot M48 to forensic DNA extraction

The development of a nucleic acid extraction method based on magnetic separation has opened up possibilities forl automation of DNA extraction. The BioRobot M48 is one of robotic stations applicable to automated DNA extraction in forensics. However, each new method should be thoroughly validated before application to routine casework. Our aim was to compare the effectiveness of the currently utilized organic/Microcon 100 based extraction procedure and magnetic extraction with BioRobot M48. The DNA concentration of DNA extracts obtained from different kinds of typical forensic material was evaluated followed by amplification with the SGM Plus or Identifiler kit and capillary electrophoresis using ABI 3100 Avant. We can conclude that BioRobot M48 is a very effective instrument for DNA extraction from most specimens and can be successfully applied in forensic laboratories.     

The Use of Hemastix® and the Subsequent Lack of DNA Recovery Using the Promega DNA IQTM System

Abstract:  Following implementation of our automated process incorporating the Promega DNA IQ^TM system as a DNA extraction method, a large number of blood-containing exhibits failed to produce DNA. These exhibits had been tested with the Hemastix^® reagent strip, commonly used by police investigators and forensic laboratories as a screening test for blood. Some exhibits were even tainted green following transfer of the presumptive test reagents onto the samples. A series of experiments were carried out to examine the effect of the Hemastix^® chemistries on the DNA IQ^TM system. Our results indicate that one or more chemicals imbedded in the Hemastix^® reagent strip severely reduce the ability to recover DNA from any suspected stain using the DNA IQ^TM magnetic bead technology. The 3,3',5,5'-tetramethylbenzidine (TMB) used as the reporting dye appears to interact with the magnetic beads to prevent DNA recovery. Hydrogen peroxide does not seem to be involved. The Hemastix^® chemistries do not interfere in any way with DNA extraction performed using phenol-chloroform. The incompatibility of the Hemastix^® chemistries on the DNA IQ^TM system forced us to adopt an indirect approach using filter paper to carry out the presumptive test.     

批量陈旧血样DNA的自动化检验

目的建立批量陈旧血样DNA自动化提取检验的方法。方法采用普通磁珠法和本文建立的磁珠法经TECAN Freedom EVO150—8型自动化工作站分别提取540份陈旧血样模板DNA,采用Sinofiler^TM试剂盒进行荧光标记复合扩增。结果采用普通磁珠法和本文所建DNA提取方法,在540份样本中获得全部基因座STR分型的样本分别为217份和488份,检验成功率分别为40．2％和90．3％。结论本文所建方法可显著提高大批量陈旧血样自动化检验成功率。     

Generating DNA profiles from immunochromatographic cards using LCN methodology

The aim of this research was to obtain DNA profiles from immunochromatographic test devices which have already yielded positive results with body fluids obtained from fourteen volunteers. Three different immunochromatographic cards for the identification of human blood and one for the identification of human saliva were used for this research. Each body fluid was detected using the appropriate immunochromatographic card. The used cards were kept at room temperature for various lengths of time. The membranes were removed at the end of the designated times and the entire strip was extracted using low copy number (LCN) extraction procedure. The extracted DNA was amplified using reduced amplification volume and higher PCR cycle numbers. Autosomal STR profiles were detected using AmpFℓSTR^® Identifiler™ PCR Amplification Kit from Applied Biosystems (AB). Additionally, DNA extracted from the male volunteers was amplified using the AB AmpFℓSTR^® Yfiler™ PCR Amplification Kit. Analysis of the amplified products was carried out by capillary electrophoresis injection on the AB 3130xl Genetic Analyzer. The generated DNA data was analyzed using the SoftGenetics GeneMarker^® HID Version 1.7 software.Autosomal and Y-STR DNA profiles were obtained from most of the cards which were stored at room temperature for up to three months. DNA profile was obtained from all four types of the immunochromatographic cards used in this study. These profiles were concordant with the profiles obtained from the donors’ reference samples.     

DNA数据库建设中批量样品不同DNA提取方法的比较

目的比较和选择自动化工作平台提取DNA的方法,并用于DNA数据库建设。方法用手工Chelex-100法、Biomek3000自动化工作平台结合Chelex-100法及DNA-IQTM磁珠法对实验室收集的建库滤纸血样进行DNA提取,荧光定量技术对上述3种方法提取的模板DNA进行测定;扩增产物用3100基因分析仪检测并用基因分析软件分析。结果手工Chelex-100法、自动化Chelex-100法及DNA-IQTM磁珠法提取的DNA模板浓度分别为0.593ng±0.131ng/μl、0.579ng±0.096ng/μl、0.447ng±0.056ng/μl;成功率分别为100%、98.9%、99.5%。结论本文建立的自动化Chelex-100法可用于大规模DNA数据库建设。     

FaSTR DNA: A new expert system for forensic DNA analysis

The automation of DNA profile analysis of reference and crime samples continues to gain pace driven in part by a realisation by the criminal justice system of the positive impact DNA technology can have in aiding in the solution of crime and the apprehension of suspects. Expert systems to automate the profile analysis component of the process are beginning to be developed. In this paper, we report the validation of a new expert system FaSTR DNA, an expert system suitable for the analysis of DNA profiles from single source reference samples and from crime samples. We compare the performance of FaSTR DNA with that of other equivalent systems, GeneMapper™ ID v3.2 (Applied Biosystems, Foster City, CA) and FSS-i³ v4 (The Forensic Science Service^® DNA expert System Suite FSS-i³, Forensic Science Service, Birmingham, UK) with GeneScan^® Analysis v3.7/Genotyper^® v3.7 software (Applied Biosystems, Foster City, CA, USA) with manual review. We have shown that FaSTR DNA provides an alternative solution to automating DNA profile analysis and is appropriate for implementation into forensic laboratories. The FaSTR DNA system was demonstrated to be comparable in performance to that of GeneMapper™ ID v3.2 and superior to that of FSS-i³ v4 for the analysis of DNA profiles from crime samples.     

Optimization of DNA extraction from low-yield and degraded samples using the BioRobot EZ1 and BioRobot M48

Robotic extraction of DNA from dilutions of blood and semen using either the BioRobots EZ1 or BioRobots M48 consistently produced lower recoveries than standard organic extractions of the same samples. In an effort to increase the efficiency of robotically extracted DNA, glycogen and carrier RNA were added following cell lysis. The addition of glycogen, postlysis, resulted in no improvement in DNA recovery with the BioRobot EZ1. However, when carrier RNA was added to the cell lysate of limited and degraded samples extracted on the EZ1 or the M48, DNA recoveries dramatically increased four- to 20-fold. DNA yields obtained by robotic extraction in the presence of carrier RNA were as high, or higher, as those obtained by organic extraction lacking carrier RNA, while experiments that utilized carrier RNA in both types of extractions showed increased sensitivity for both methods. Furthermore, carrier RNA substantially increased the recovery of fragmented DNA with the EZ1.     

Successful validation of a fully automated sample lysis workstation

The Netherlands Forensic Institute recently validated a fully automated system for sample lysis, the AutoLys workstation and AutoLys tubes, developed in collaboration with Hamilton Robotics. We have carried out the developmental validation of the AutoLys system and concluded it offers a solution to drastically reduce user-errors, increase DNA yields, gain in productivity, while maintaining quality. This article reports on the study setup and discusses the results of comparing the AutoLys workstation to the conventional (manual) method. The AutoLys system proved to successfully process all commonly found forensic sample types, producing high quality extracts, containing on average 12.1% more DNA than manually extracted samples (as determined by qPCR and STR-profiling). The optimized robotic protocol minimizes the risk on cross-contamination (no cross-contamination detected in over 500 samples tested) and retains the flexibility to adjust chemically relevant variables specific to a laboratory's individual requirements. The fully automated process enables complete sample-tracking, drastically reducing the risk on user-errors. The NFI is currently validating an on-deck magnetic bead-based purification of the lysates.     

Implementation of a semi-automated processing system for DNA profiling of forensic casework samples

The concept for a semi-automated processing system for DNA analysis of crime scene samples was developed at the Landeskriminalamt Baden-Württemberg (LKA BW) and comprises the extraction of genomic DNA from human cells by ChargeSwitch^® magnetic bead technology (CST), quantification of purified DNA by real-time PCR, amplification of short tandem repeats (STRs) by PCR and DNA fragment length analysis of STRs by capillary electrophoresis. Three liquid handling workstations from Tecan, a real-time PCR device and a 16-channel capillary electrophoresis (CE) system, both from Applied Biosystems (AB), are linked via laboratory data network. Transmission and management of sample and analysis data is enabled by a Laboratory Information and Management System (LIMS). Suitability for a wide range of stain types, early exclusion of DNA-free samples, barcode sample identification and prevention of cross-contaminations guarantee efficiency and high quality standards.     

改良醋酸铵盐析法提取福尔马林固定石蜡包埋组织DNA

目的采用改良醋酸铵盐析法提取福尔马林固定石蜡包埋组织DNA,并评价其应用价值。方法取死后24h内人体肾组织用10%中性福尔马林溶液固定7d,石蜡包埋。采用酚/氯仿法、Chelex-100法及改良醋酸铵盐析法提取DNA。经用紫外分光光度计法、AGE及PCR-PAGE检测比较不同方法提取DNA的质量。结果改良醋酸铵盐析法、酚/氯仿法、Chelex-100法OD260/OD280比值分别为1.962±0.195、2.110±0.470、1.018±0.124,两两比较均有显著性差异(P<0.05);3种方法提取DNA含量(μg)分别为0.515±0.447、0.328±0.345、5.346±1.994;AGE电泳图谱可印证上述结果;PCR-PAGE检测显示改良醋酸铵盐析法提取DNA的谱带清晰度好于其它两种方法。结论改良醋酸铵盐析法更适合微量福尔马林固定石蜡包埋组织样本DNA的提取。     

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.     

Comparison of five DNA quantification methods

Six commercial preparations of human genomic DNA were quantified using five quantification methods: UV spectrometry, SYBR-Green dye staining, slot blot hybridization with the probe D17Z1, Quantifiler™ Human DNA Quantification kit and RB1 rt-PCR. All methods measured higher DNA concentrations than expected based on the information by the manufacturers. UV spectrometry, SYBR-Green dye staining, slot blot and RB1 rt-PCR gave 39, 27, 11 and 12%, respectively, higher concentrations than expected based on the manufacturers’ information. The DNA preparations were quantified using the Quantifiler™ Human DNA Quantification kit in two experiments. The measured DNA concentrations with Quantifiler were 125 and 160% higher than expected based on the manufacturers’ information. When the Quantifiler™ human DNA standard (Raji cell line) was replaced by the commercial human DNA preparation G147A (Promega) to generate the DNA standard curve in the Quantifiler™ Human DNA Quantification kit, the DNA quantification results of the human DNA preparations were 31% higher than expected based on the manufacturers’ information. The results indicate a calibration problem with the Quantifiler™ human DNA standard for its use with the Quantifiler™ Human DNA Quantification kit. The possible reasons for the problem are discussed and a solution is suggested. The results emphasise the need for standard reference DNA material and standard methods for DNA quantification.