Direct amplification of STRs from blood or buccal cell samples

Forensic databasing laboratories routinely analyze blood or buccal cell samples deposited on FTA^® paper. Prior to PCR amplification of the STRs, the FTA^® samples must undergo multi-step sample purification protocols to remove the PCR inhibitors present within the sample and from the FTA^® paper. The multi-step sample purification protocols are laborious, time-consuming and increase the potential for sample cross-contamination.To eliminate the need for DNA purification, we conducted studies to optimize the PCR buffer and thermal cycling parameters to allow for direct amplification of STRs from blood or buccal samples on FTA^® paper. We evaluated the effect of various factors on the DNA profile including: FTA^® disc size, blood sample load variation, and buffer formulation. The new STR assay enables the direct amplification of DNA from single source samples on FTA^® discs without sample purification. The new STR assay improves the workflow by eliminating tedious steps and minimizing sample handling. Furthermore, the new STR assay reduces cost by eliminating the need for purification reagents and expensive robots.     

荧光标记STR分型技术检验腐败组织基因型

探讨腐败组织荧光标记STR分型检测技术的应用价值。应用含12个STR基因座及一个性别基因座的2个荧光标记的复合扩增系统,对40例1～6周的腐败肌肉提取的DNA进行扩增,用变性聚丙烯酰胺凝胶电泳,PE377测序仪分析基因型。所检测样本在12个STR基因座均扩增出特异性谱带,并可判定其基因型。荧光标记STR检测技术对腐败组织分型可靠,在实际检案中具有较高的应用价值。     

全基因组扩增在微量检材DNA分型中的应用

目的探索全基因组扩增技术对微量检材DNA分型的有效性。方法通过显微操作制备含1~20个细胞的模拟微量检材样本,在常规PCR-STR分型前加入全基因组扩增步骤,从等位基因不平衡、等位基因丢失、基因座丢失、伪等位基因(包含stutter峰)等方面探究PEP和MDA两种全基因组扩增方法对微量检材DNA分型的有效性。结果 MDA扩增效率高于PEP,但等位基因丢失和伪等位基因严重;PEP方法的正确分型率高于MDA,但小片段DNA优势扩增现象较严重。结论 MDA方法并不适合目前以STR分型为主导的法庭科学,当微量检材样本的绝对量相当少时,可以考虑使用PEP方法来扩大样本量,以满足重复检验的要求,但可能面临大片段DNA扩增失败的风险。     

荧光STR直接复合扩增试剂缓冲体系的研制

目的研制适用于数据库样本荧光STR直接复合扩增体系。方法针对常规血卡、FTA和903血卡样本,配制扩增缓冲液基准母液,采用不同配方的扩增缓冲体系进行直接扩增及检测。考察不同种类增强剂、4种商业化DNA聚合酶、不同复性温度和终延伸时间对检材的检测效果,并验证优化体系的适应性。结果采用本文所建体系对各类血卡样本进行检验,均可获得样本清晰、完整的STR分型。体系选择BSA\Tween20\DMSO\甘油等增强剂组合、Typer热启动聚合酶1.5U/10μL、57～59℃复性温度、30～50min终延伸时间,采用10μL体系即可对直径1.2mm FTA卡血样进行有效分型。结论本文所研制的缓冲体系能够满足常规血卡、FTA和903血卡样本直接扩增检验的需要。     

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.     

Northeast Italy population data using multiplex PCR (HUMCD4, HUMTH01, HUMTPOX, and HUMCSF1P0) loci

Allele and genotype frequencies for four short tandem repeat (STR) loci (HUMCD4, HUMTH01, HUMTPOX, and HUMCSF1P0) were determined in 100 unrelated individuals from Veneto (Northeast Italy). After a multiplex polymerase chain reaction (PCR)-amplification, semi-automatic DNA profiling was performed using an A.L.F.express DNA Sequencer. Conditions were optimized for the PCR co-amplification of these four STR loci and the quadruplex PCR was performed on various forensic DNA samples such as whole blood, blood-stains, teeth, and saliva from Caucasians living in the Northeast Italy. The distribution of the genotype frequencies showed no significant deviation from Hardy-Weinberg expectations in the sampled population. The combined Power of Discrimination (PD) of the quadruplex was 0.9999.     

Development and validation of the AmpFlSTR MiniFiler PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA

DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpFlSTR MiniFiler PCR Amplification Kit has been developed. The kit contains reagents for the amplification of eight miniSTRs which are the largest sized loci in the AmpFlSTR Identifiler PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpFlSTR SGM Plus kit. This informative nine-locus multiplex, which includes the gender-identification locus Amelogenin, has been validated according to the FBI/National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpFlSTR Identifiler and SGM Plus kits. These data support that the MiniFiler kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.     

A time course study on STR profiles derived from human bone, muscle, and bone marrow.

The use of the polymerase chain reaction (PCR) to define deoxyribonucleic acid (DNA) types at several loci was investigated. PCR was used to amplify nine short tandem repeat (STR) loci along with the amelogenin locus on the X and Y chromosomes using the AmpF/STR Profiler Plus PCR amplification kit (Perkin Elmer). Rib bones were collected from 12 individuals. Five cm portions were buried at a depth of approximately 30 cm and 5 cm portions were left on the surface of the ground. Samples were exposed to the environment for periods of time ranging from two weeks to 17 months. Dried blood standards were prepared for use as reference standards for each rib sample. Bone, muscle, and bone marrow were collected from each sample. DNA from each tissue type was extracted. Complete profile results were obtained from the surface bone samples out to an exposure time of 17 months. None of the muscle or bone marrow samples produced complete profile results beyond eight weeks. All DNA typing results from complete or incomplete profiles were consistent with DNA typing results of the corresponding blood standard. Results suggest that using the AmpF/STR Profiler Plus PCR Amplification Kit is a valid way to establish the DNA profile of tissue types from human remains.     

Identification of a novel polymorphism in the X-chromosome region homologous to the DYS456 locus

During an extensive multipopulation study with Y-short tandem repeat (STR) loci, amplified using the AmpFlSTR Yfiler PCR amplification kit, amplification of a 71 bp fragment was observed in 2.32% of the male samples analyzed (N = 3141). By direct sequencing of this fragment, it was determined that the primer binding sequences were identical to those of the DYS456 locus. A T to G single-nucleotide polymorphism (SNP) enabled amplification of the 71 bp fragment. The SNP is located within an X-Y homologous region at Xq21.31 and was observed with the highest frequency within the African American and Sub-Saharan African populations in our study. Presence of SNP on the X chromosome did not interfere with the reliability of typing the DYS456 locus and the other Y-STR loci typeable using the AmpFlSTR Yfiler PCR amplification kit. Full profiles in a mixture of male:female at 1:4000 were obtained using the current configuration of the AmpFlSTR Yfiler kit even in the presence of female DNA containing the G variant.     

Use of “AnyDirect PCR buffer” for PCR amplification of washed bloodstains: A case report

AnyDirect PCR buffer (BioQuest) permits to perform direct PCR from different kinds of forensic samples (blood, saliva, sperm, etc.) without any DNA purification step.This is very useful in particular when working with small quantity of sample since it avoids the risk of loosing sample step by step as often happens with traditional DNA extraction procedures.In the present casework we analyzed some bloodstains found by luminol test onto washed clothes and linen: all samples were analyzed either using the AmpFlSTRs Identifiler kit (Applied Biosystems).     

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。     

汗潜指印的STR分型检测在案件中的应用

目的探索案例中所涉及的汗潜指印DNA的提取和检验方法。方法采用Chlex-100法提取DNA,进行STR复合扩增,通过毛细管电泳检测荧光信号。结果案例中涉及的汗潜指印在ProfilerPlus试剂盒10个基因座的分型检测均获成功。结论含有汗潜指印的检材的发现和正确提取对最终检测成功至关重要。     

Validation of short tandem repeats (STRs) for forensic usage: performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples

The amplification and typing conditions for the 13 core CODIS loci and their forensic applicability were evaluated. These loci are CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11. Results were obtained using the multiplex STR systems AmpFlSTR Profiler Plus and AmpFlSTR COfiler (Applied Biosystems, Foster City, CA), GenePrint PowerPlex (Promega Corporation, Madison, WI), and subsets of these kits. For detection of fluorescently labeled amplified products, the ABI Prism 310 Genetic Analyzer, the ABI Prism 377 DNA Sequencer, the FMBIO II Fluorescent Imaging Device, and the Fluorlmager were utilized. The following studies were conducted: (a) evaluation of PCR parameter ranges required for adequate performance in multiplex amplification of STR loci, (b) determination of the sensitivity of detection of the systems, (c) characterization of non-allelic PCR products, (d) evaluation of heterozygous peak intensities, (e) determination of the relative level of stutter per locus, (f) determination of stochastic PCR thresholds, (g) analysis of previously typed case samples, environmentally insulted samples, and body fluid samples deposited on various substrates, and (h) detection of components of mixed DNA samples. The data demonstrate that the commercially available multiplex kits can be used to amplify and type STR loci successfully from DNA derived from human biological specimens. There was no evidence of false positive or false negative results and no substantial evidence of preferential amplification within a locus. Although at times general balance among loci labeled with the same fluorophore was not observed, the results obtained were still valid and robust. Suggested criteria are provided for determining whether a sample is derived from a single source or from more than one contributor. These criteria entail the following: (a) the number of peaks at a locus, (b) the relative height of stutter products, and (c) peak height ratios. Stochastic threshold levels and the efficiency of non-templated nucleotide addition should be considered when evaluating the presence of mixtures or low quantity DNA samples. Guidelines, not standards, for interpretation should be developed to interpret STR profiles in cases, because there will be instances in which the standards may not apply. These instances include (a) a primer binding site variant for one allele at a given locus, (b) unusually high stutter product, (c) gene duplication, and (d) translocation.     

Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes

Abstract:  With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus? STR reactions alone and in combination with AmpliTaq^® Gold. All reactions included the additional step of a post‐PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.     

印记基因KCNQ1的遗传多态性及在亲权鉴定中的应用

目的为了调查印记基因KCNQ1的STR位点在中国汉族人群中的遗传多态性，利用亲源印记等位基因（parentally imprinting allele，PIA）分型法确定孩子的等位基因亲代来源，为亲权鉴定提供新的侯选STR位点。方法应用Chelex法提取153例佳木斯地区汉族健康无血缘关系个体DNA，用QIAamp Blood Kit（Qiagen）法提取3个家庭10个个体DNA，PCR扩增，凝胶电泳分型，ABIPRISM^TM 3730XL DNA测序仪测序；甲基化敏感性限制性内切酶消化孩子基因组DNA，PCR扩增，确定孩子等位基因的亲代来源。结果发现在中国佳木斯地区汉族人群中KCNQ1基因的STR有7个等位基因，多态信息含量为0．662，且KCNQ1基因的STR位点呈父源印记。结论印记基因KCNQ1的STR位点有很好的多态性，可为亲权鉴定提供新的侯选遗传标记，其亲源特异性甲基化标记有望应用于单亲鉴定中。     

微腔型PCR芯片用于法医DNA分析的初步研究

目的为了克服传统PCR热循环仪体积大,运行电压高,耗时长,只能在实验室中应用的缺点,研究了一种微腔型PCR芯片,以期实现现场对STR片段的复合扩增。方法采用在PCR反应缓冲液中加入不同浓度的BSA溶液对芯片进行表面优化处理的方法及不同酶量优化实现对STR片段的有效扩增。结果使用浓度为0．5mg／mL的BSA可得到清晰完整的STR分型结果;加大酶量有益于扩增效率的提高。结论该种微腔型PCR芯片经初步优化后可有效地对STR片段进行复合扩增,经进一步优化可真正实现法医DNA分析的更加微量化和快速化。     

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).     

Simplified low-copy-number DNA analysis by post-PCR purification

Frequently, evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Such low-copy-number (LCN) samples are usually subjected to increased amplification cylces to obtain genetic data. In this study, a 28-cycle polymerase chain reaction (PCR) was used to evaluate various methods of post-PCR purification for their effects on the sensitivity of fluorophore-based allelic detection subsequent to capillary electrophoretic separation. The amplified product was purified using filtration, silica gel membrane, and enzyme mediated hydrolysis purification techniques and evaluated for their effect on fluorescent allelic signal intensity. A purification method was selected and its effect on fluorescent allelic signal intensity was compared with that of the unpurified PCR product. A method of post-PCR purification is described which increases the sensitivity of standard 28-cycle PCR such that profiles from LCN DNA templates (<100 pg DNA) can be obtained. Full DNA profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele dropout, increased stutter, and sporadic contamination typical of LCN analysis were observed; however, no contamination was observed in negative amplification controls. Post-PCR purification of the PCR product can increase the sensitivity of capillary electrophoresis to such an extent that DNA profiles can be obtained from <100 pg of DNA using 28-cycle amplification.     

Evaluation of Direct PCR Amplification Using Various Swabs and Washing Reagents

DNA profiles were generated via direct amplification from blood and saliva samples deposited on various types of swab substrates. Each of the six non-FTA substrates used in this research was punched with a Harris 1.2 mm puncher. After 0.1 μL of blood or 0.5 μL saliva, samples were deposited on each of these punches, samples were pretreated with one of four buffers and washing reagents. Amplification was performed using direct and nondirect autosomal and Y-STR kits. Autosomal and Y-STR profiles were successfully generated from most of these substrates when pretreated with buffer or washing reagents. Concordant profiles were obtained within and between the six substrates, the six amplification kits, and all four reagents. The direct amplification of substrates which do not contain lysing agent would be beneficial to the forensic community as the procedure can be used on evidence samples commonly found at crime scenes.     

Estimating the probability of allelic drop-out of STR alleles in forensic genetics

In crime cases with available DNA evidence, the amount of DNA is often sparse due to the setting of the crime. In such cases, allelic drop-out of one or more true alleles in STR typing is possible. We present a statistical model for estimating the per locus and overall probability of allelic drop-out using the results of all STR loci in the case sample as reference. The methodology of logistic regression is appropriate for this analysis, and we demonstrate how to incorporate this in a forensic genetic framework.