The Optimization of Electrophoresis on a Glass Microfluidic Chip and its Application in Forensic Science

Microfluidic chips offer significant speed, cost, and sensitivity advantages, but numerous parameters must be optimized to provide microchip electrophoresis detection. Experiments were conducted to study the factors, including sieving matrices (the concentration and type), surface modification, analysis temperature, and electric field strengths, which all impact the effectiveness of microchip electrophoresis detection of DNA samples. Our results showed that the best resolution for ssDNA was observed using 4.5% w/v (7 M urea) lab‐fabricated LPA gel, dynamic wall coating of the microchannel, electrophoresis temperatures between 55 and 60°C, and electrical fields between 350 and 450 V/cm on the microchip‐based capillary electrophoresis (μCE) system. One base‐pair resolution could be achieved in the 19‐cm‐length microchannel. Furthermore, both 9947A standard genomic DNA and DNA extracted from blood spots were demonstrated to be successfully separated with well‐resolved DNA peaks in 8 min. Therefore, the microchip electrophoresis system demonstrated good potential for rapid forensic DNA analysis.     

Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

Psilocybe cubensis, or “magic mushroom,” is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web‐based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy^® Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR^® Green I, Radiant? Green, or LCGreen Plus^® intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR^® Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant? Green dye.     

Simultaneous Identification of Four “Legal High” Plant Species in a Multiplex PCR High‐Resolution Melt Assay,

The international prevalence of “legal high” drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real‐time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high‐resolution melt using LCGreen Plus^®. The PCR assay was evaluated based on the following: (i) specificity and selectivity—primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity—serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability—sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging “legal high” species.     

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.     

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.     

A Simple Method of VNTR D1S80 Locus Allelic Ladder Construction for Capillary Electrophoresis‐based Genotyping

VNTR D1S80 locus genotyping has been largely replaced in forensics by STR. As the statute of limitations on murder cases was abolished in the Republic of Korea in July 2015, the demand for re‐analysis of DNA from unresolved murder cases has increased. The National Forensic Service includes several recorded D1S80 genotypes as crucial clues. Here, we re‐established the D1S80 analysis system using capillary electrophoresis and confirmed the reproducibility of the system by comparison with the genotypes of eight DNA samples that had been analyzed using PAGE in 2006. In addition, we created an allelic ladder via new methodology using flanking region sequences. A single DNA sample (K562) and seven primers were used for the new ladder, which contains 12 alleles. Although artificial owing to the use of the flanking region rather than repeat unit reduction, the method is rapid and simple, and could be applicable in any laboratory.     

Optimization and validation of a fast amplification protocol for AmpFlSTR Profiler Plus for rapid forensic human identification

The goal of this work was to optimize and validate a fast amplification protocol for the multiplex amplification of the STR loci included in AmpFlSTR^® Profiler Plus^® to expedite human DNA identification. By modifying the cycling conditions and by combining the use of a DNA polymerase optimized for high speed PCR (SpeedSTAR™ HS) and a more efficient thermal cycler instrument (Bio-RAD C1000™), we were able to reduce the amplification process from 4 h to 26 min. No modification to the commercial AmpFlSTR^® Profiler Plus^® primer mix was required. When compared to the current Royal Canadian Mounted Police (RCMP) amplification protocol, no differences with regards to specificity, sensitivity, heterozygote peak height ratios and overall profile balance were noted. Moreover, complete concordance was obtained with profiles previously generated with the standard amplification protocol and minor alleles in mixture samples were reliably typed. An increase in n − 4 stutter ratios (2.2% on average for all loci) was observed for profiles amplified with the fast protocol compared to the current procedure. Our results document the robustness of this rapid amplification protocol for STR profiling using the AmpFlSTR^® Profiler Plus^® primer set and demonstrate that comparable data can be obtained in substantially less time. This new approach could provide an alternative option to current multiplex STR typing amplification protocols in order to increase throughput or expedite time-sensitive cases.     

用于DNA分离的毛细管电泳无胶筛分介质研究进展

毛细管电泳是一种在生命科学应用最为广泛的生物分子分离技术,目前已广泛应用于DNA测序,SNP检测,DNA片段分析等。筛分介质是毛细管电泳性能的关键因素,筛分介质的种类、结构、分子量及所形成的溶液决定着生物分子的迁移特性、分辨率、读出长度、重现性等参数,对分离结果有重大影响。本文对近年来用于DNA分离的无胶筛分介质的研究和应用情况进行综述,希望能对相关研究和鉴定提供参考和借鉴。     

A comparison of three automated DNA purification methods in Forensic casework

Manual Chelex^®-100 and organic extractions (phenol/chloroform) are used as routine methods at the Swedish National Laboratory of Forensic Science, SKL. The aim of this study was to find an automated DNA purification system to replace the organic method. The following methods were evaluated and compared to each other and to the organic method used routinely; BioRobot^® EZ1 with EZ1 DNA Investigator Kit and Card (Qiagen), iPrep™ Purification Instrument with iPrep™ ChargeSwitch^® Forensic Kit and Card (Invitrogen), Magnatrix™ 1200 Workstation with the Magnatrix™ gDNA Blood Kit Forensic and two different protocols; Forensic protocol A and B (Magnetic Biosolutions). Blood on fats, cotton swabs, moist snuff, paper towels and leather, post-mortem blood and muscle tissue were extracted with the different methods. DNA concentration and quality of the electropherograms were examined. Individual comparisons between the four extraction methods showed that iPrep™ and Magnatrix™ 1200 gave significantly lower mean quantities compared to BioRobot^® EZ1 and the organic extraction method (p < 0.05). There were no significant differences between the latter two. BioRobot^® EZ1 generated the best results and is in the process of being validated for routine analysis at SKL.     

短串联重复位点ACTBP2(SE33)的扩增片段长度多态性研究

应用变性聚丙烯酸胺凝胶电泳（dn－PAGE）结合银染色技术对短串联重复（STR）位点ACTBP2（SE33）的扩增片段长度多态性（Amp－FLPs）进行了研究。在210名无关中国个体中观察到了25个等位基因，等位基因频率分布在0．007～0．093之间。基因型的分布符合Hardy－Weinberg定律，个体识别能力（DP）值为0．99，杂合度（H）为98．7％。七个家系分析的结果表明，该位点的遗传符合孟德尔遗传法则，未观察到变异。对几种常见的法医物证检材的分析表明，该分型系统对DNA降解放为严重的检村适用性强，而且灵敏度高（0．5ng），适合于法医学实际应用。     

Validating TrueAllele® Interpretation of DNA Mixtures Containing up to Ten Unknown Contributors

Most DNA evidence is a mixture of two or more people. Cybergenetics TrueAllele^® system uses Bayesian computing to separate genotypes from mixture data and compare genotypes to calculate likelihood ratio (LR) match statistics. This validation study examined the reliability of TrueAllele computing on laboratory-generated DNA mixtures containing up to ten unknown contributors. Using log(LR) match information, the study measured sensitivity, specificity, and reproducibility. These reliability metrics were assessed under different conditions, including varying the number of assumed contributors, statistical sampling duration, and setting known genotypes. The main determiner of match information and variability was how much DNA a person contributed to a mixture. Observed contributor number based on data peaks gave better results than the number known from experimental design. The study found that TrueAllele is a reliable method for analyzing DNA mixtures containing up to ten unknown contributors.     

Developmental validation of the AmpF?STR SEfiler Plus™ PCR amplification kit: An improved multiplex with enhanced performance for inhibited samples

Since its introduction in 2002, the AmpF?STR^® SEfiler™ kit has provided a highly discriminating DNA profiling option to German forensic laboratories by combining the widely used SGM Plus^® Kit loci with the SE-33 locus required for the German DNA Database. Whilst proving successful on database samples, laboratories using the SEfiler™ kit have reported the need for chemistry better able to handle the ever-increasing number of casework samples.The new AmpF?STR^® SEfiler Plus™ kit contains the same loci and primer sequences as the SEfiler™ kit but uses improved synthesis and purification processes to minimize the presence of dye-labeled artifacts. Other improvements include modified PCR cycling conditions for enhanced sensitivity and a new buffer formulation that improves performance with inhibited samples when compared to the original SEfiler™ kit.Validation studies demonstrating the effectiveness of the multiplex are presented with emphasis on the models of inhibition and casework samples.     

Validation of a 17-locus Y-STR multiplex system

Internal validation of a commercial 17-locus Y-STR system (AmpFlSTR^® Yfiler™, Applied Biosystems) has been performed on the ABI PRISM^® 3130 Genetic Analyzer for use in forensic cases. The Yfiler™ kit was validated according to SWGDAM guidelines. Our results show that it is possible to obtain full profiles with as little as 30 pg of male DNA even in the presence of 20,000-fold amounts of female DNA. Reaction volume was optimized for 10 μl. Male-male mixtures yielded full profiles of the minor contributor with 10-fold excess of the major contributor. Stutter values for each locus were determined from data generated for the population study which included Y-STR profiles from 156 caucasian males from the Montreal and Lac St.-Jean areas of Québec, Canada. The study recorded 141 different haplotypes of which 131 were unique with a haplotype diversity of 0.9965. A number of non-probative forensic samples from rape kit epithelial fractions and fingernail scrapings were also successfully tested.     

Mitochondrial DNA Validation in a State Laboratory*,†

Abstract: Because of the inception of the FBI Regional mitochondrial DNA (mtDNA) laboratories, many do not see establishing state/local mtDNA processing laboratories as a priority. Yet there is a long‐term need for mtDNA processing that will exceed the capabilities of the FBI Regional mtDNA laboratories and the few other laboratories that are currently processing mtDNA, and that need can be fulfilled by state/local laboratories. Thus, the DNA Unit of the Delaware Office of the Chief Medical Examiner (OCME‐DNA Unit) completed validation of in‐house mtDNA testing in January 2007. The validation plan for mtDNA processing included the following sections: preliminary research, sensitivity and contamination studies, ExoSAP‐IT^® optimization, BigDye^® optimization, sequencing and 310 optimization, sample preparation and extraction optimization, heteroplasmy, mixtures, and reproducibility. All sections of the validation were successfully completed, and mtDNA processing of skeletal remains, teeth, and hairs, as well as blood and buccal reference samples was adopted by the OCME‐DNA Unit.     

Internal validation of 29 autosomal SNP multiplex using a ABI 310 genetic analyser

In the last few years genetic identification and paternity testing have begun to make increasing use of autosomal SNP (Single Nucleotide Polymorphism) typing as a supplement or alternative to STR analysis. With the improvement in detection technology SNP analysis is likely to be easier and more sensitive, with the generation of new methods and multiplex systems for a growing array of SNP markers. SNPforID consortium developed 52 SNP PCR multiplex for human identification purposes detected with 23 plex and 29 plex single base extension reactions (Auto1 and 2 respectively). In this study, internal validation for the 29 SNPs of Auto2 was carried out by performing a 29 plex PCR and single base extension reaction on control samples and previously analyzed forensic casework and subsequent detection with an AB 310 Genetic Analyzer. We tested the accuracy, precision, sensitivity and reproducibility of the Auto2 multiplex with this instrument in our laboratory. We used 9947A control DNA samples of the AmpF?STR Identifiler™ kit to test the validation parameters together with non-probative DNA samples from whole blood and buccal swab samples of 29 healthy donors from different parts of Istanbul. Good results were obtained but interpretation of the peak patterns obtained on the AB 310 requires care and thorough optimization before they can be readily compard to those obtained from multiple capillary AB 31xx Analyzers. We succesfully optimized and validated the SNPforID Auto2 multiplex system for identification analyses in our laboratory.     

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.     

Evaluation of sieving polymers for fast, reproducible electrophoretic analysis of short tandem repeats (STR) in capillaries

Efficient capillary electrophoretic STR analysis requires rapid, reproducible and robust separation of DNA fragments with reasonable capillary longevity--this is currently accomplished using proprietary commercial polymeric sieving matrices specifically developed for this separation. These matrices, while effective, are costly and do not provide adequate resolution of STR DNA fragments in capillaries with shorter effective separation lengths, increasing the time required to accomplish the separation and minimizing the potential extrapolation to other miniaturized platforms. As the forensic community looks toward next generation microchip technology as a means of processing casework more rapidly, new sieving polymers need to be evaluated for utilization in this platform. The research presented here describes the assessment of commercially-available polymeric sieving matrices for STR analysis, with consideration given to feasibility of incorporation into a microdevice. Polymer composition, molecular weight, and concentration were evaluated, along with an assessment of the effects of buffer composition, separation temperature, and capillary length. These variables were evaluated individually or collectively on the ability to resolve STR DNA fragments and the reproducibility of the separations and the results compared to a proprietary commercial product. A 600,000 Da MW poly(ethylene oxide) (PEO) solution at a 3% (w/v) concentration was determined to be the most suitable matrix for these separations. This polymer, in coated capillaries, provided highly robust and reproducible separations, with near baseline resolution of fragments having single base differences. Reductions in the temperature of the separation, from 60 degrees C to 40 degrees C, and the urea concentration of the buffer, from 7 M to 3.5 M, provided increased longevity of the PEO polymer for repeated separations. Comparison of this polymer with currently specified commercial products used for STR analysis showed that the optimized PEO matrix provided superior separations under all conditions tested. In addition, PEO could be utilized in shorter capillary systems, with a concurrent decrease in analysis time, highlighting its potential for use in shortened capillary or microdevice systems.     

Internal Validation of RapidHIT®ID ACE Sample Cartridge and Assessment of the EXT Sample Cartridge*†

A new rapid DNA solution, the RapidHIT^®ID, can accommodate two different sample cartridges, ACE, for the analysis of a single swab and EXT, for the analysis of DNA extracts. An efficient internal validation designed for low‐throughput rapid DNA is described. An evaluation of the EXT sample cartridge is also described. Each cartridge generated profiles with sufficient data quality to meet CODIS eligibility in fewer than 120 min. The results exhibited 100% correlation when compared to conventional DNA typing methods. Precision, reproducibility, stochastic, mixture, and contamination experiments produced expected results. Sensitivity of the ACE sample cartridge was acceptable for buccal swab analysis. The sensitivity of the EXT sample cartridge is discussed. The ACE validation and the EXT evaluation utilized a minimalist, cost‐saving, efficient design to generate a validated RapidHIT^®ID instrument capable of producing genetic profiles from both extracted forensic DNA samples and buccal swab samples within 120 min.     

Evaluation of the Differex™ System

Differential extraction is an efficient method to separate sperm cells from epithelial cells. A manual Chelex^®-100 based method is used at the Swedish National Laboratory of Forensic Science, SKL. The Differex™ System (Promega) uses a Proteinase K digestion of epithelial cells followed by centrifugation and phase separation. The sperm- and epithelial fractions are further purified with DNA IQ™ System (Promega) or with phenol/chloroform. The Differex™ System in combination with DNA IQ™ System were evaluated and compared to the Chelex^®-100 method used routinely. After modifications, the Differex™ System gave comparable results to the Chelex^®-100 method. The modifications included additional Proteinase K and DTT, longer incubation time and additional steps when removing the solid support from the Digestion Solution. In the Chelex^®-100 based method microscopic examination is done on the sperm pellet in a total volume of 50 μl. It was not possible to do a microscopic examination in less than 100 μl using the Differex™ System. Additionally the sperms were in clusters of epithelial cell debris. Microscopic examination is an important part of the differential extraction at SKL. Therefore, the Differex™ System will not be implemented at our laboratory.     

Evaluation of a multicapillary electrophoresis instrument for mitochondrial DNA typing

Laser-induced detection of fluorescent labeled PCR products and multi-wavelength detection (i.e., multicolor analysis) enables rapid generation of mtDNA sequencing profiles. Traditionally, polyacrylamide slab gels have been used as the electrophoretic medium for mtDNA sequencing in forensic analyses. Replacement of slab gel electrophoresis with capillary electrophoresis (CE) can facilitate automation of the analytical process. Automation and high throughput can be further enhanced by using multicapillary electrophoretic systems. The use of the ABI Prism 3100 Genetic Analyzer (ABI 3100, Applied Biosystems, Foster City, CA) as well as the ABI Prism 310 Genetic Analyzer (ABI 310, Applied Biosystems, Foster City, CA) were evaluated for mtDNA sequencing capabilities and compared with sequencing results obtained on the platform currently in use in the FBI Laboratory (the ABI Prism 377 DNA Sequencer, ABI 377, Applied Biosystems, Foster City, CA). Various studies were performed to assess the utility of the ABI 3100, as well as the ABI 310 for mtDNA sequencing. The tests included: comparisons of results obtained among the ABI 3100, the ABI 310 and the ABI 377 instruments; comparisons of results obtained within and between capillary arrays; evaluation of capillary length; evaluation of sample injection time; evaluation of the resolution of mixtures/heteroplasmic samples; and evaluation of the sensitivity of detection of a minor component with reduced template on the ABI 3100. In addition, other studies were performed to improve sample preparation; these included: comparison of template suppression reagent (TSR, Applied Biosystems, Foster City, CA) versus formamide; the use of Performa DTR Gel Filtration Cartridges (Edge BioSystems Inc., Gaithersburg, MD) versus Centri-Sep Spin Columns (Princeton Separations, Adelphia, NJ) for product purification after cycle sequencing; and sample stability after denaturation. The data support that valid and reliable results can be obtained using either capillary electrophoresis instrument, and the quality of sequencing results are comparable to or better than those obtained from the ABI 377 instrument.