Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection

Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full Identifiler™ profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm.     

AMELY无效扩增个体Yp11.2区域STS位点检测

目的 通过Y-STR复合扩增技术检测AMELY缺失的男性个体Y染色体的完整性,并对数据库中的STS位点进行筛选并设计引物,检测Y染色体AMEL区域的STS位点缺失情况,为进一步研究中国人口中AMELY的缺失提供数据和理论依据.方法 应用Goldeneye 20A PCR扩增试剂盒、华夏TM白金PCR扩增试剂、Y fil...     

直接扩增法在血痕、肋软骨和唾液检材中的应用

目的采用Identifiler Direct PCR试剂盒直接扩增法进行棉签擦拭血痕、肋软骨和烟蒂唾液斑DNA分型检验,并评价其应用价值。方法收集棉签擦拭血痕、烟蒂各20份,肋软骨10份,采用Identifiler Direct PCR试剂盒进行直接扩增及分型检验,以相同检材采用磁珠法/Chelex-100法提取模板DNA后扩增检验结果作为对照,对两组所得结果进行比较分析。结果棉签擦拭血痕和肋软骨一次检测完整分型率均为100%,分型结果与对照组一致;烟蒂上唾液斑有2份检材第一次未能完整分型,调整方法再次检验后获分型成功。结论实际检案中的棉签血痕、肋软骨和烟上唾液斑,采用直接扩增法检测,方法简单、快速、稳定、检材用量小,可在实际检案中选择使用。     

Fixation of cells for analysis by laser microdissection--comparative studies in forensic trace material

This paper is focused on the preparation of samples for laser microdissection (LM) in forensic casework. In forensic genetics, it is essential to preserve and separate cellular traces during sample preparation, as they are usually gathered in very small amounts and are often contaminated with undesired cells. This is made possible by laser microdissection, a technique developed to cut cells or tissue of a certain type from a microscopical specimen by UV laser and catapult them directly into a PCR reactor. This method minimizes the risk of getting inconclusive, mixed DNA profiles due to contamination by foreign DNA and also supplies information about the cellular origin of a DNA profile. A method for optimized fixation and staining of spermatozoa for laser microdissection was established. Four different fixation methods combined with two staining methods were tested on two different microscope slides. Moreover, the effect of a blocker pen to contain the specimen on the slide was investigated.     

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。     

Efficiency Analysis of EX16 +10Y Kit on Detection of the Uygur Population in Xinjiang ProvinceCSCD

Objective: To analyse the efficiency of EX16+10Y kit on the forensic detection of the Uygur in Xinjiang province. Methods: The blood samples were extracted from 4 620 male individuals of Uygur in Xinjiang province, and amplified by EX16+10Y kit. The typing of amplification products was performed by 3130xl genetic analyzer. Results: The genotyping graphs of 15 autosomal STR loci and 10 Y -chromosomal STR loci from 4 620 male individuals of Uygur in Xinjiang province were acquired completely. The genotype distribution of 15 autosomal STR loci was consistent with Hardy-Weinberg equilibrium. The heterozygosity, polymorphism information content and discrimination power of STR loci were 0.637-0.838, 0.580-0.860 and 0.811-0.978, respectively. There were 766 haplotypes in 10 Y -chromosomal STR loci. Conclusion: The test results of EX16+10Y kit is accurate and trustworthy, which can simultaneously be used for the individual identification and the screening of paternal pedigree in practical work. © 2018 by the Editorial Department of Journal of Forensic Medicine.     

Reduction of Powerplex® Y23 Reaction Volume for Genotyping Buccal Cell Samples on FTATM Cards

PowerPlex^® Y23 is a novel kit for Y‐STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y‐STR analysis. This study examined PCR amplification from 1.2‐mm FTA punch in reduced volumes of 5 and 10 μL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 μL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High‐quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 μL, respectively, in the first attempt. Volume reduction to 5 μL has a vast economic impact especially for DNA database laboratories.     

Difficulties of sex determination from forensic bone degraded DNA: A comparison of three methods

Sex determination is of paramount importance in forensic anthropology. Numerous anthropological methods have been described, including visual assessments and various measurements of bones. Nevertheless, whatever the method used, the percentage of correct classification of a single bone usually varies between 80% and 95%, due to significant intra- and inter-population variations, and sometimes variations coming from secular trends. DNA is increasingly used in a forensic context. But forensic DNA extraction from bone raises several issues, because the samples are very often badly altered and/or in very small quantity. Nuclear DNA is difficult to get from degraded samples, according to low copy number, at least in comparison with mitochondrial DNA. In a forensic context (as in a paeleoanthropological context) DNA sex determination is usually complicated by the weak amount of DNA, the degraded nature of nucleic acids, the presence of enzymatic inhibitors in DNA extracts, the possible faint amplification of Y band and the risk of contamination during either excavation or manipulation of samples.The aim of this work was to compare three methods of DNA sex determination from bones: procedure #1 using a single PCR amplification, procedure #2 using a double PCR amplification, and procedure #3 adding bleaching for decontamination of the bone, instead of simply rubbing the bone. These processes were applied to samples of bones (49 samples coming from 39 individuals) that were in various states of post mortem alteration.The main results are the following. (i) No DNA could be extracted from three skulls (parietal bones, mastoid process), the compact bone of one rib, and the diaphysis of one femur; (ii) there was a contamination in three skulls; and (iii) the Y band did not appear in two male cases, with one of the three procedures (male tibia, procedure #2) and with procedures #2 and #3 (male femur).This study emphasises the main issue while working with altered bones: the impossibility to extract DNA in some cases, and, worth of all, the contamination of the sample or the faint amplification of Y band which leads to a wrong sex answer. Multiple and significant precautions have to be taken to avoid such difficulties.     

激光捕获显微切割技术用于分离混合斑中精子细胞

目的 评估激光捕获显微切割(laser capture microdissection.LCM)技术在分离混合斑中少量精子细胞的应用价值.方法 配制不同比例的精液-阴道上皮细胞混合液.分别用差异裂解法和LCM法分离精子细胞,用磁珠法提取精子细胞DNA,并用IdentifilerTM试剂盒进行STR基因型检测.结果 LC...     

不同保存时间和不同精子数量精斑样本DNA分型比较

目的比较不同保存时间和不同精子数量精斑样本DNA分型的效果。方法制备精斑样本,保存10d的样本采用激光显微捕获30、20、15、10、5、1个精子,用于不同数量精子分型比较;保存10d、214d、375d的样本分别捕获30、20、10个精子,用于不同保存时间分型比较。比较各组检出率、等位基因丢失率和非特异性扩增率,采用χ2检验进行差异比较。结果①不同精子数量分型：捕获10个精子即可得到完整的DNA分型,且随着精子数增多,检出率逐渐提高而等位基因丢失率逐渐降低,30个精子等位基因丢失率为0%,1个精子则可达58.89%;②不同保存时间分型：总趋势是保存时间越短,捕获精子越多检出率越高,10个精子与20、30个精子组比较,均有显著性差异（P〈0.05）;等位基因丢失率及非特异性扩增率则随保存时间的延长而增加,相同保存时间的不同精子数量组之间和相同的精子数量的不同保存时间组之间比较,差异均具有统计学意义（P〈0.05）。结论激光显微捕获精子数目和检材保存时间对DNA分型结果有直接影响。     

Effectiveness of Coupled Application of AmpFℓSTR Yfiler Kit and Reduced Size Y‐chromosomal Short Tandem Repeat Analysis for Archeological Human Bones

The AmpF?STR Yfiler PCR Amplification (Yfiler) kit continues to be improved for a better analytical efficiency in cases of highly degraded DNA. The authors endeavored to determine whether coupling of the Yfiler kit with supplemental multiplex amplification of some Y‐STR loci is a more efficient analytical mode for poorly preserved human femurs (n = 15) discovered at Korean archeological sites. To reveal locus profiles not easily obtained by Yfiler analysis, custom‐designed primers were adopted for the DYS390, DYS391, DYS392, DYS438, DYS439, and DYS635 loci. The success rate for 16 Y‐STR locus profiles obtained from the 15 femurs was improved from 18.33% (in the use of Yfiler kit only) to 49.17% (the coupled use of Yfiler and custom‐designed primers). In this study, the authors established that the custom‐designed primers offer a markedly improved success rate for obtainment of Y‐STR profiles from degraded aDNA not easily identified by sole use of the Yfiler assay.     

荧光原位杂交技术在法庭科学DNA检验中的应用

目的运用荧光原位杂交技术结合激光显微切割技术,分离法医物证男女混合样本中的男性细胞和女性细胞,并进行DNA分型。方法通过双色荧光原位杂交,Y染色体标记上绿色信号,X染色体标记上红色信号,在荧光显微镜下识别男性细胞和女性细胞,并通过激光显微切割技术分别获得男性细胞和女性细胞进行DNA分型。结果运用荧光原位杂交技术,能够分别标记法医物证男女混合样本中的男性细胞和女性细胞,并通过激光显微切割技术获得各自的DNA分型。结论荧光原位杂交技术结合激光显微切割技术,可应用于法医物证男女混合样本的检验,提高个体识别能力。     

Autosomal short tandem repeat analysis of ancient DNA by coupled use of mini- and conventional STR kits

Abstract: Multiplex autosomal short tandem repeat (STR) genotyping enables researchers to obtain genetic information from ancient human samples. In this study, we tested newly developed AmpF?STR^® MiniFiler? kit for autosomal STR analysis of ancient DNA (aDNA), using human femurs (n = 8) collected from medieval Korean tombs. After extracting aDNA from the bones, autosomal STR analyses were repeated for each sample using the AmpF?STR^® MiniFiler? and Identifiler? kits. Whereas only 21.87% of larger‐sized loci profiles could be obtained with the Identifiler? kit, 75% of the same loci profiles were determined by MiniFiler? kit analysis. This very successful amplification of large‐sized STR markers from highly degraded aDNA suggests that the MiniFiler? kit could be a useful complement to conventional STR kit analysis of ancient samples.     

Y-STR analysis on DNA mixture samples—Results of a collaborative project of the ENFSI DNA Working Group

The ENFSI (European Network of Forensic Science Institutes) DNA Working Group undertook a collaborative project on Y-STR typing of DNA mixture samples that were centrally prepared and thoroughly tested prior to the shipment. Four commercial Y-STR typing kits (Y-Filer, Applied Biosystems, Foster City, CA, USA; Argus Y Nonaplex, Biotype, Dresden, Germany; Powerplex Y, Promega, Madison, WI, USA; and DYSplex-3, SERAC, Bad Homburg, Germany) were used for the amplification of the mixture samples. The results of the study showed a striking inter-laboratory difference of kit performance as determined from the peak heights of the obtained Y-STR genotypes. Variation in quantity and quality of the shipped DNA can be excluded as reason for the observed differences because both samples and shipping conditions were found to be reproducible in an earlier study. The results suggest that in some cases a laboratory-specific optimization process is indicated to reach a comparable sensitivity for the analysis of minute amounts of DNA.     

Population data for Y-chromosome haplotypes defined by 17 STRs (AmpFlSTR YFiler) in Central Portugal

The 17 Y-chromosome STR loci (DYS19, DYS389I, DYS389II, DYS390, DYS456, DYS391, DYS392, DYS393 and DYS385 a/b, DYS458, DYS439, DYS635, GATA H4.1, DYS437, DYS438 and DYS448) were determined for 100 unrelated males, living in Central Portugal, using the AmpFlSTR YFiler PCR Amplification kit (Applied Biosystems).A total of 99 different haplotypes were found, with only two individuals sharing the same haplotype. The overall haplotype diversity (HD) was determined as 0.9998, a value similar to other Y Filer data sets.Y-STR polymorphisms in Central Portugal population, using YFiler, provide a powerful discrimination tool for routine forensic applications.     

Internal Validation of the AmpFlSTR Yfiler™ Amplification Kit for Use in Forensic Casework

Abstract:  Y-chromosomal short-tandem repeat (Y-STR) amplification has been used in forensic casework at the Bureau of Criminal Apprehension (BCA) Forensic Science Laboratory since 2003. At that time, two separate amplifications were required to type the SWGDAM recommended loci (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438, and DYS439). The Yfiler™ kit coamplifies these loci as well as DYS437, DYS448, DYS456, DYS458, DYS635, and Y GATA H4. The Yfiler™ kit was validated following the internal validations outlined in the SWGDAM revised validation guidelines. Our studies show that 0.125 ng of male DNA will generate a complete 17 locus profile and that as little as 0.06 ng of male DNA yields an average of nine loci. In the male–male mixtures, a complete profile from the minor component was detected up to 1:5 ratio; most of the alleles of the minor component were detected at a 1:10 ratio and more than half the alleles of the minor component were detected at a 1:20 ratio. Complete YSTR profiles were obtained when 500 pg male DNA was mixed with female DNA at ratios up to 1:1000. At ratios of 1:5000 and 1:10,000 (male DNA to female DNA) inhibition of the YSTR amplification was evident. The YSTR results obtained for the adjudicated case samples gave significantly more probative information than the autosomal results. Our studies demonstrate that the Yfiler™ kit is extremely sensitive, does not exhibit cross-reactivity with female DNA, successfully types male DNA in the presence of overwhelming amounts of female DNA and is successful in typing actual forensic samples from adjudicated cases.     

Internal validation of the AmpFlSTR Yfiler amplification kit for use in forensic casework.

Y-chromosomal short-tandem repeat (Y-STR) amplification has been used in forensic casework at the Bureau of Criminal Apprehension (BCA) Forensic Science Laboratory since 2003. At that time, two separate amplifications were required to type the SWGDAM recommended loci (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438, and DYS439). The Yfiler kit coamplifies these loci as well as DYS437, DYS448, DYS456, DYS458, DYS635, and Y GATA H4. The Yfiler kit was validated following the internal validations outlined in the SWGDAM revised validation guidelines. Our studies show that 0.125 ng of male DNA will generate a complete 17 locus profile and that as little as 0.06 ng of male DNA yields an average of nine loci. In the male-male mixtures, a complete profile from the minor component was detected up to 1:5 ratio; most of the alleles of the minor component were detected at a 1:10 ratio and more than half the alleles of the minor component were detected at a 1:20 ratio. Complete YSTR profiles were obtained when 500 pg male DNA was mixed with female DNA at ratios up to 1:1000. At ratios of 1:5000 and 1:10,000 (male DNA to female DNA) inhibition of the YSTR amplification was evident. The YSTR results obtained for the adjudicated case samples gave significantly more information than the autosomal results. Our studies demonstrate that the Yfiler kit is extremely sensitive, does not exhibit cross-reactivity with female DNA, successfully types male DNA in the presence of overwhelming amounts of female DNA and is successful in typing actual forensic samples from adjudicated cases.     

Standard 28-cycle amplification of X/Y-FISH labelled epithelial cells for DNA profiling

The examination of sexual assault evidence frequently involves the analysis of samples that comprise mixtures of male and female cells. Separating male and female cells benefits analysis as the results are more likely to be simplified into profiles from single contributors. Some separation methods have focussed on separation of sperm from epithelial cells, but samples without sperm also require separation (vasectomised males, licked skin, etc.). X/Y chromosome FISH labelling when combined with laser micro-dissection (LMD) is a reliable method to separate male and female epithelial cells, but has mostly been combined with increased cycle PCR to create DNA profiles, limiting its use in many forensic laboratories. This study aimed to determine the limits of cell numbers collected by LMD for standard 28-cycle DNA profiling, and to test the effects, if any, on stochastic variation normally caused by sampling effects. Male and female epithelial cells were stained using the Vysis CEP X/Y DNA Probe kits, and collected using a Leica LMD6000. DNA was extracted and amplified by the ESR in-house one-tube method, using standard 28-cycle PCR with the AmpFISTR Identifiler™ (Applied Biosystems) multiplex kit. Full IdentifilerTM DNA profiles were produced using standard 28-cycle PCR, and partial profiles suitable for submission were produced from even relatively low numbers of cells collected. Profiling results were compared with low-copy number PCR on low numbers of cells stained and collected in the same manner, and the observed effects on heterozygote balance are discussed.     

Research status and prospects of DNA test on difficult specimens

This paper reviews the advances of DNA detection on three types of difficult biological specimens including degraded samples, trace evidences and mixed samples. The source of different samples, processing methods and announcements were analyzed. New methods such as mitochondrial test system, changing the original experimental conditions, low-volume PCR amplification and new technologies such as whole genome amplification techniques, laser capture micro-dissection, and mini-STR technology in recent years are introduced.     

26个Y-STR基因座遗传多态性及突变调查

目的 调查26个Y-STR基因座的突变率和遗传多态性,研究其法医学应用效能.方法 本文以575对蒙古族父子对为模板,统计26个Y-STR基因座的突变率,并且研究26个Y-STR基因座在黑龙江省蒙古族、江西省汉族及福州市汉族等3个地区777个无关男性个体中的遗传多态性,评估该试剂盒的法医学应用价值.结果 26个Y-STR...