Quantification of human mitochondrial DNA using synthesized DNA standards

Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust.     

The utility of whole genome amplification for typing compromised forensic samples

Biological evidence has become invaluable in the crime laboratory; however, it may exist in limited quantity and/or quality. Given this, the ability to amplify total DNA obtained from evidence, in an unbiased manner, would be highly advantageous. Methods for whole genome amplification (WGA) have the potential to fulfill this role, resulting in a virtually unlimited supply of DNA. In the research presented, two WGA methods, improved primer extension preamplification and multiple displacement amplification (MDA), were tested using commercial kits. Control DNA, artificially degraded DNA, and DNA from fresh blood, aged blood, hair shafts, and aged bones underwent WGA, followed by short tandem repeat and mitochondrial DNA analysis. The methods did amplify DNA, but performed poorly on forensically relevant samples; the maximum amplicon size was reduced, and MDA often resulted in extraneous bands following polymerase chain reaction. Taken together, WGA appears to be of limited forensic utility unless the samples are of a very high quality.     

Quadruplex real-time PCR for forensic DNA quantitation

Forensic DNA quantitation is an important initial step preceding PCR amplification of the STR loci even though information concerning the quality of the DNA is not revealed. A quadruplex real-time PCR (qPCR) assay was developed to quantify four DNA targets: (1) the human RB1 gene in nuclear DNA, (2) the DAZ gene present on the human Y chromosome, (3) the ATPase8 gene present in human mitochondrial DNA and (4) an artificial internal positive control to reveal possible PCR inhibition. Primers labeled with four different fluorophores are used together with a single quencher using the antiprimer quenching-based qPCR method in one reaction, in which the resultant amplicons are less than 127 bp in size. Sensitivity was shown to be less than ten copies for all four targets in the absence of amplification inhibition. The amplification remained sensitive in the presence of an excess of non-human DNA.     

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability.     

DNA Typer~(TM)15 plus直扩试剂盒在DNA数据库建设中的应用

目的测试DNA TyperTM15 plus直扩试剂盒的技术性能指标,评价其在DNA数据库建设中的应用价值。方法采用DNA TyperTM15 plus试剂盒,并使用IdentifilerTM和DNA TyperTM15试剂盒进行比较,设定不同体系和引物量、不同退火温度和循环次数以进行方法验证;设定不同模板量标准品、不同比例混合样本,取猪、狗、兔等动物的血液样品,血痕、骨骼、唾液斑等常见检材样本以及不同建库样本,以验证试剂盒灵敏度、特异性、稳定性以及混合样本、常见检材及建库样本的检测能力。结果直扩试剂盒分型结果准确,重复性好,灵敏度可达0.125ng,不同批次间试剂检测结果稳定,对不同检材有很好的适应性。10μL扩增体系时FTA卡和加强型血液采集卡取样直径应为0.5mm,而血滤纸、血液采集卡样本和经典型血液采集卡取样直径应为1.0mm。结论 DNA TyperTM15 plus直扩试剂盒的性能可以满足DNA数据库建设及检案的需要,可在相关实验中选择使用。     

Comparison of two whole genome amplification methods for STR genotyping of LCN and degraded DNA samples

The analysis of LCN or highly degraded DNA samples presents a challenge for forensic science. Improving the quantity and/or quality of samples would greatly increase the profiling success rate from LCN and degraded samples. Whole genome amplification (WGA) is one method that has such potential. Two commercially available WGA kits, GenomePlex and GenomiPhi, were investigated for use on LCN and degraded DNA samples. Both kits amplified genomic DNA, producing microgram quantities from sub-nanogram templates. Profiling success of LCN DNA samples was increased, with improvements of over 700% from 10pg template DNA compared to non-WGA-amplified control samples. The amplification success with degraded DNA was also improved by WGA. Degraded DNA was simulated using restriction enzymes to demonstrate that the application of WGA can result in the typing of STR loci that could not previously be amplified. An increase in artefacts, such as stutter alleles and amplification biases, were observed in many samples. Results show that WGA is capable of increasing both the quality and quantity of DNA, and has the potential to improve profiling success from difficult samples in forensic casework.     

低拷贝模板DNA分析技术研究进展

近年来,低拷贝（LCN）模板类生物物证在法医DNA分析中占有了越来越重要的地位。用于低拷贝模板DNA的检测方法也得到飞速发展。本文通过对各种LCN-DNA分析技术如增加扩增循环数、纯化扩增产物、全基因组扩增、激光捕获显微切割等检测方法的综述,以及对LCN—DNA检测结果的分析评价,全面介绍LCN分析技术在法庭科学应用的最新进展及存在问题。最大限度地拓展低拷贝模板类生物物证在刑事司法领域的应用。     

受污染烟蒂的DNA检验

采用chelex-100提取DNA并加以纯化,对受泥土等物严重污染的烟蒂中唾液上皮细胞DNA进行PCR扩增分型,成功地应用于案件检验,该方法方便,实用.     

GoldeneyeTM 20A-M试剂盒在数据库建设中的应用

目的 研究GoldeneyeTM 20A-M试剂盒在DNA数据库建设中的应用.方法 针对血滤纸特性,优化不同条件血痕样品的取样量、扩增循环次数,缩短PCR扩增时间,以建立适合批量样品直接快速扩增检验的方法,并将其应用于1 200份数据库样品的检验中.结果 确定了批量样本检验的最适取样量和扩增循环次数,建立了快速扩增反应程序,1 200份样品的直接快速扩增检验成功率为98.4％,批量检验92份样品(1板)从取样到电泳完成只需6h.结论 GoldeneyeTM 20A-M试剂盒的应用,免去DNA提取过程,方法简单、快速、获得信息量大,适用于DNA数据库建设的需要.     

线粒体16srRNA和ND4基因在种属鉴定中的应用研究

目的构建一种用于种属鉴定的线粒体DNA(m tDNA)16 srRNA和ND4基因荧光标记复合扩增检测体系。方法利用引物设计软件(Prim er 5)对两个m tDNA序列ND4基因和16 srRNA基因设计两对引物,每对引物中的一条在5’端标记荧光素(6-FAM)。按传统复合扩增技术建立复合扩增体系,用AB I PR ISM 310基因分析仪对产物进行分析。结果人类DNA扩增产物出现两个峰,片段大小分别为110bp的人类特异片段和149bp的人与动物共有片段,而动物DNA扩增产物出现一个峰,片段大小为149bp。对30个实验室存放5~15年的陈旧人血痕也能明确判断其种属来源。结论该体系可以明确区分人源性生物检材与其它常见动物样本,对实验室长期存放的陈旧检材也具有较好的检测能力。     

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.     

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.     

多重置换扩增技术用于法医学微量DNA检测效果

目的探讨多重置换扩增(MDA)技术对法医学微量DNA样品STR检测分型的效果。方法用MDA技术对不同模板量DNA进行全基因组扩增(WGA),扩增产物用实时荧光定量PCR技术定量、用Profiler PlusTM试剂盒检测基因型。结果该方法可对模板DNA增加104～106倍。1ng样品DNA的MDA产物可获得9个STR基因座和Amelogenin性别基因座的准确分型结果;低于0.1ng的样品DNA经MDA扩增后,基因座检出数增加,但可见等位基因不平衡或丢失现象。结论MDA技术可有效增加DNA模板量和提高微量DNA分型效果。但样品DNA量低于0.1ng时,MDA产物的STR分型结果判读须慎重。     

A Study of PCR Inhibition Mechanisms Using Real Time PCR*,†

Abstract: In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co‐extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance.     

The Role of DNA‐Stained Currency in Gang Robberies within the United Kingdom

Cash‐and‐valuables‐in‐transit (CViT) robberies have become a substantial problem—especially in the current global economy. Over £19.4 million were stolen in 2008 and £17.1 million in 2009 in the United Kingdom alone. The transportation of cash and valuable items between financial institutions has long been a target of robberies in the U.K. After a robbery has occurred, police collect evidence in the form of ink‐stained currency. These stained notes are submitted for analysis. Samples are subjected to polymerase chain reaction in order to amplify any possible botanically derived DNA markers present on the notes. After amplification, capillary electrophoresis allows for the deciphering of the “DNA profile.” The DNA profile is then linked to a particular cash box, and this information is compared with records of whether or not that box had been stolen. The cases below are three such instances where botanically marked currency was used to help solve robberies.     

Forensic touch DNA recovery from metal surfaces – A review

Trace evidence such as touch (also known as contact) DNA has probative value as a vital forensic investigative tool that can lead to the identification and apprehension of a criminal. While the volume of touch DNA evidence items submitted to forensic laboratories has significantly increased, recovery and amplification of DNA from these items, especially from metal surfaces, remains challenging. Currently little is understood with regards to the underlying mechanisms of metal-DNA interactions in the context of forensic science and how this may impact on DNA recovery. An increased understanding of these mechanisms would allow optimisation of methods to improve outcomes when sampling these materials. This paper reviews the basis of DNA binding to metal substrates, the merits and limitations of current methods and future perspectives of improving recovery and amplification of touch DNA from metal surfaces of forensic interest.     

全基因组扩增技术及其在法医遗传学中的应用前景

全基因组扩增技术是近年来发展起来的新型PCR技术,它提供了一种从微量基因组DNA获取大量遗传信息的途径,为法医处理微量检材提供了一个有用的工具。本文综述了全基因组技术及其种类和原理和其在法医遗传学方面的应用前景。     

Rapid amplification of commercial STR typing kits

Forensic DNA typing is currently conducted in approximately 8–10 h. The process includes DNA extraction, quantitation, multiplex PCR amplification, and fragment length detection. Today's commercial multiplex short tandem repeat (STR) typing kits are not optimized for rapid PCR thermal cycling. Current protocols require approximately 3 h for amplifying a multiplex containing 15 STR loci plus amelogenin. With the continuing development of miniaturization technologies such as microfluidic and micro-capillary devices, there is a desire to reduce the overall time required to type DNA samples. Such miniature devices could be used for initial screening at a crime scene, at a border, and at airports. There is also the benefit of reducing the required PCR amplification time for labs typing single-source reference samples. Surveys of fast processing polymerases working in combination with rapid cycling protocols have resulted in the development of a ‘rapid’ PCR amplification protocol. Results are obtained in less than 36 min run on a standard peltier-based thermal cycler employing a heating rate of 4 °C/s. Capillary electrophoresis characterization of the PCR products indicates good peak balance between loci, strong signal intensity and minor adenylation artifacts. Genotyping results are concordant with standard amplification conditions utilizing a standard 3 h (non-rapid) thermal cycling procedure. The rapid assay conditions are robust enough to routinely amplify 0.5 ng of template DNA (with 28 cycles).     

The Effects of Different Maceration Techniques on Nuclear DNA Amplification Using Human Bone

Abstract: Forensic anthropologists routinely macerate human bone for the purposes of identity and trauma analysis, but the heat and chemical treatments used can destroy genetic evidence. As a follow‐up to a previous study on nuclear DNA recovery that used pig ribs, this study utilizes human skeletal remains treated with various bone maceration techniques for nuclear DNA amplification using the standard Combined DNA Index System (CODIS) markers. DNA was extracted from 18 samples of human lower leg bones subjected to nine chemical and heat maceration techniques. Genotyping was carried out using the AmpF?STR^® COfiler^® and AmpF?STR^® Profiler Plus^® ID kits. Results showed that heat treatments via microwave or Biz/Na₂CO₃ in sub‐boiling water efficiently macerate bone and produce amplifiable nuclear DNA for genetic analysis. Long‐term use of chemicals such as hydrogen peroxide is discouraged as it results in poor bone quality and has deleterious effects on DNA amplification.     

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.