Deoxyribonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs in Japanese.

The deoxyribonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs is described. HLA-DQA1 genotypes could be determined from single plucked hair roots. However, it was not easy to type HLA-DQA1 with hair shaft portions. Increase in the specimens of hair shaft portions (over 10 cm in length) to get sufficient DNA caused inhibition of polymerase chain reaction (PCR). Synthetic melanin as well as the one extracted from hairs inhibited the PCR of the genomic DNA template when added to the PCR reaction at the concentrations over than 15 ng/100 microL. Therefore, typability of hair shaft portions seems to depend on the delicate balance of the concentrations of DNA and the contaminated melanin in the final DNA extracts.     

用引物Y_3,Y_4和DNA扩增技术作血液(痕)和毛根性别鉴定的研究

作者用引物Y_3、Y_4和DNA聚合酶链式反应(PCR)作微量人类血液(痕)和毛根的性别鉴定。扩增的靶序列位于Y染色体DNA特异3.4kb重复序列中,扩增产物为460bp。检材用量为:新鲜血液0.5μl、血痕纱纤维1mm、毛根单个。20例保存4个月的血痕与2例保存6年半的血痕性别判定结果均正确,无性别记载的保存9～11年的3例血痕显现了清晰的460bpY特异DNA扩增带。15例保存20天的自然脱落毛根性别判定结果均正确。本法省略了检材处理中的酚-氯仿抽提DNA等纯化步骤,既简化了实验操作,又减少了检验过程中外源DNA的污染机会和样品DNA的损耗,使这一性别鉴定方法更符合法医学实践的需要。     

Enzyme typing of human hair roots.

Sufficient phosphoglucomutase activity was found to be present in plucked hair noses bearing either fragmentary or complete outer root sheaths to enable typing of individual roots by starch-gel electrophoresis. Hair roots collected by brushing were found to contain very little PGM activity. Other isoenzyme systems were detected in hair roots but in insufficient quantities to make typing feasible.     

Sex determination from plucked human hairs without epithelial root sheath

For sex determination, it is difficult to examine the sex chromatin in the hair root devoid of the epithelial root sheath because of melanin deposition in the hair cortex cells. In the present study, the possibility of determining sex was sought by observing the frequency of Y chromatin in the nuclei of hair cortex cells from human hair root devoid of epithelial root sheath. The male hair contained nuclei in each of which one brightly fluorescent spot was recognizable. The average frequency of the fluorescent spot in the hair cortex nuclei observed in 90 samples from 30 males was 59.4%; the maximum frequency was 74% and the minimum 37%. On the other hand, the fluorescent spots were rarely found in the nuclei of hair cortex cells from female hair. The frequency of occurrence was 5.3% on average, the maximum frequency being 12%, the minimum 0%. The frequency distribution of the male group and female group exhibited two discontinuous peaks. The frequency of the fluorescent spots can therefore be used as a criterion for determining sex. The fluorescent spot in dried male hair was shown to be detectable in a frequency of 20 – 40% 4 weeks after plucking, indicating that the nuclei of hair cortex cells can be subjected to the examination for at least this period if the hair has been kept in a dry condition.     

Sex determination from plucked human hairs without epithelial root sheath. II. Depigmentation of melanin in the hair cortex before Feulgen reaction

In hair roots devoid of the epithelial root sheath, an attempt was made to decolorize the melanin granules without affecting the Feulgen reaction for the sex chromatin. The hair samples were treated with 0.25% potassium permanganate for 1 hour, 0.3% hydrogen peroxide for 1 minute and 5% oxalic acid for 5 minutes, and then stained with Feulgen. The frequency of sex chromatins ranged from 22% to 47% (average 32%) in female samples and from 0% to 8% (average 5%) in male samples. Thus, the frequency distributions of the male and female samples were completely independent of each other. The sex chromatins in dried female hairs were detectable at a frequency of 16 – 26% several weeks after plucking. The depigmentation procedure almost completely bleached the melanin granules in the hair cortex, and produced no harmful effect on the Feulgen reaction that followed.     

用引物Y_3、Y_4和PCR方法鉴定性别的法医学应用

用Y3、Y4和Alu9．1、Alug．2两对引物和PCR方法检测陈旧血痕和毛根的性别获得成功。引物Y3、Y4扩增的靶序列位于Y染色体特异3．4Kb重复序列中，扩增产物为460bp；引物Alu9．1、Alu9.2用以扩增男女共有的Alu重复序列，扩增产物为130bP。室温保存13年之久的19例脐带血血痕（男性9冽，女性10例）和室温保存10～11个月的10例已知性别自然脱落毛根（男性6例，女性4例）的性别测定结果均正确；对一起凶杀案的血痕性别测定为定案提供了重要证据。本方法简化了样品的前处理过程。     

用PCR扩增X-Y-特异片段性别鉴定的研究

采用5%Chelex-100处理方法提取 DNA,用 PCR 扩增 Amelogenin 基因片段检测血痕和毛根的性别。扩增一个样品只需1/10体积3mm~2血痕 Chelex-100处理液或1/10体积一根毛发 Chelex-100处理液(含模板 DNA)。100例血液样品结果显示,在男性可同时显现977bpX 特异片段和788bpY 特异片段,而在女性只观察到977bp X 特异片段,该方法用于性别鉴定可靠、灵敏、方便。     

一种用于降解DNA样本的性别鉴定方法

目的　建立一种采用PCR技术对降解DNA样本进行性别鉴定的新方法。　方法　采用针对amelogenin基因X染色体外显子 3bp缺失设计的引物AMELU1及AMELD1,对在室温环境下放置 5～ 15年的男、女血痕标本各 5 0例、毛发各 2 0例、骨骼各 2 0例以及现场提取 5 - - 2 0天的男、女腐败肌肉各 10例标本中提取的降解DNA样本进行扩增。用PAG( 9%T ,3 %C)电泳、银染显带检测扩增产物。　结果　所有样本均得到正确结果 ,男性检材表现为 83bp的Y特异性及 80bp的X特异性 2条谱带 ,而女性检材仅有 1条 80bp的X特异性谱带。　结论　用针对amelogenin基因X染色体外显子 3bp缺失设计的引物AMELU1及AMELD1鉴定性别的方法灵敏、可靠、方便 ,是降解DNA检材性别鉴定十分理想的方法。     

考古样品中Amelogenin同源基因的提取和检测

目的利用人类性染色体Amelogenin同源基因在X、Y染色体上序列长度的差异,选择设计引物,对考古样本进行古DNA性别信息研究。方法采用苯酚/氯仿-二氧化硅-超滤离心方法提取东岭墓葬群殉人骨骼、牙齿古DNA,PCR扩增,非变性聚丙烯酰胺凝胶电泳(PAGE)分离和检测古DNA扩增片段。结果8个墓葬16个样品中有7个样品出现阳性扩增检测,目标基因片段清楚,男性为二条带(X、Y),女性为一条带(X),牙齿样品检测成功率优于骨骼样品。结论改进的苯酚/氯仿—二氧化硅—超滤分离法是较好的古DNA提取方法,具有降低PCR抑制剂、消耗成本低和提取成功率高等特点。基于人类X、Y染色体Amelogenin同源基因的古DNA性别分析方法可成为分子考古重要的技术方法。     

PCR－RFLP技术鉴定ABO基因型的法医学应用研究

目的建立PCR－RFLP、非变性PAG胶垂直电泳和银染技术进行ABO基因分型的方法体系,并对200名广东汉族人群ABO基因型频率进行了调查。方法用Chelex－100和酚、氯仿抽提法处理样本,PCR扩增后用非变性聚丙烯酰胺凝胶垂直电泳和银染技术检测分型。结果ABO位点特异性扩增片段长度为175bp～210bp,6种基因型频率分布为0.0250～0.4300,杂合度H值为0.5162,个体识别力DP值为0.7111。结论该方法可成功运用于血液、血痕、精斑、毛发、骨组织和混合斑等检材的个体识别及亲权鉴定的检验。     

Evaluation of usefulness of further Y-STR analysis in sexual assault cases on PSA positive samples resulting in female autosomal STR profiling

Samples from male to female sexual assault cases that are positive for the presumptive prostate specific antigen (PSA) test often do not result in a male autosomal STR profile. Due to highly unequal proportions of female and male DNA in typical sexual assault samples, routine autosomal STR analysis often fails to detect the DNA of the assailant, even after differential extraction of the samples. Previous studies have already shown the value of Y-STR analysis in such cases [1]. In Belgium, forensic DNA laboratories are only allowed to perform Y-STR profiling on sexual assault samples by a specific requisition, after routine autosomal STR analysis has been performed. However, a request for additional Y-STR analysis is rather exceptional.In this study, we evaluated the usefulness of further Y-STR analysis. For 100 PSA positive rinses and swabs from male to female sexual assault cases resulting in female autosomal STR profiling, 7% resulted in a full or partial Y-STR profile useful for comparison, using the 23-loci Y-STR PowerPlex Y23 System (Promega). The success rate raised to 12.5% with a higher DNA input in the PCR mix. In conclusion, these results support the usefulness of performing Y-STRs analysis on the sperm DNA extracts to identify the alleged assailant in sexual assault cases.     

Identification of male bloodstains by dot hybridization of human Y chromosome-specific deoxyribonucleic acid (DNA) probe

The sex determination of bloodstains was performed using a human Y chromosome-specific (DNA) fragment of 1.9-kb length as a hybridization probe. The DNA samples were taken from 1- and 4-week-old bloodstains of males and females, respectively. Strong signals with male DNA were observed by Y-probe, while faint signals with female DNA were detected. In addition, clear signals were observed in the extract samples from male bloodstains (16-week-old) on paper. Dot hybridization of the Y-probe would be widely applicable to studies on sex determination of medicolegal materials such as blood, bloodstains, teeth, and cadaverous parts.     

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.     

Nuclear and mitochondrial DNA quantification of various forensic materials

Due to the different types and quality of forensic evidence materials, their DNA content can vary substantially, and particularly low quantities can impact the results in an identification analysis. In this study, the quantity of mitochondrial and nuclear DNA was determined in a variety of materials using a previously described real-time PCR method. DNA quantification in the roots and distal sections of plucked and shed head hairs revealed large variations in DNA content particularly between the root and the shaft of plucked hairs. Also large intra- and inter-individual variations were found among hairs. In addition, DNA content was estimated in samples collected from fingerprints and accessories. The quantification of DNA on various items also displayed large variations, with some materials containing large amounts of nuclear DNA while no detectable nuclear DNA and only limited amounts of mitochondrial DNA were seen in others. Using this sensitive real-time PCR quantification assay, a better understanding was obtained regarding DNA content and variation in commonly analysed forensic evidence materials and this may guide the forensic scientist as to the best molecular biology approach for analysing various forensic evidence materials.     

Multiplexing of Y chromosome specific STRs and performance for mixed samples

A combination of four Y-specific polymorphic STR loci was amplified simultaneously using fluorescently labeled primers. Multiplex conditions required optimization to eliminate constant bands and amplification products for female DNA. A series of experiments was carried out for mixtures of DNA from two males, and from male and female individuals for the Y-specific STRs and an autosomal locus. For the male/male mixtures amplified with the Y specific system, and amplified for an autosomal locus, the minor component in the mixture could only be identified up to a ratio of 1:10, 1:50 respectively. In male/female DNA mixtures the Y STR alleles could be identified for the highest ratio tested, 400 pg male in DNA in 800 ng female DNA which amounts to a ratio of 1:2000.     

Mitochondrial DNA amplification success rate as a function of hair morphology

This study examines the amplification success rate of mitochondrial DNA from human head hair with respect to their potential for forensic application. Mitochondrial DNA was isolated using a Chelex-based extraction method and amplified using the LINEAR ARRAY duplex PCR system. The particular focus of this study was to characterize the morphological features of human head hair in order to further the understanding of the factors that influence amplification success rate in hair tissue using the LINEAR ARRAY duplex PCR system. 2554 head hairs from 132 individuals representing four population groups were amplified. The hair samples were characterized as follows: 1251 were identified microscopically as telogen hairs and 1303 were classified as hairs without roots (removed before extraction). Amplification success was assessed as a function of several independent variables: morphological characteristics; telogen root versus no root; donor age; scalp origin; use of cosmetic hair treatments; and race of the donor. The results show that a positive correlation exists between amplification success and the presence of a telogen root. Combining the amplification success with either the original or optimized protocol, telogen hairs result in an overall success rate of 77.5% compared with 65% for hairs with no roots. Controlling for telogen hairs, the findings indicate that the overall success rate is independent of cosmetic hair treatments; medulla structure; shaft length, diameter, and volume; and scalp origin. Conversely, the age of the donor, the race of the donor, and hair pigmentation all contribute to a variation in amplification success rate.     

Variation in nuclear DNA concentrations during urination

This study examined the cellular origin and concentration of nuclear DNA in human urine. Ten subjects provided two entire, first-morning voids: one as a single specimen and one as a consecutive series of samples. The serial samples were centrifuged, organically extracted, and quantified by slot-blot analysis. Total DNA concentrations ranged from 0.02 to 21.3 ng/mL for the males and 25.0 to 96.9 ng/mL for the females. The female samples were found to contain numerous vaginal epithelial cells. DNA was detected in all of the serial samples of nine subjects; however, the DNA concentrations varied considerably. With six subjects, the DNA concentration of the first serial sample was at least three times greater than that of the entire void. DNA was only detected in the first 21% of the void from one male subject. The results of this study have implications for the collection of urine samples.     

An innovative transfer DNA experimental design and qPCR assay: Protocol and pilot study

Forensic “touch” DNA samples are low-quantity samples that are recovered from surfaces that have been touched by single or multiple individuals. These samples can include DNA from primary contributors who directly touched the surface, as well as secondary contributors whose DNA was transferred to the surface through an intermediary. It is difficult to determine the type of transfer, or how often and under what conditions DNA transfer occurs. In this paper, we present an innovative protocol that combines (1) a paired male and female transfer DNA experimental design in which the presence of male DNA indicates secondary transfer and (2) a cost-effective quantitative PCR (qPCR) assay of a sex-specific region in the Amelogenin gene to detect male and female DNA. We evaluate the ability of the Amelogenin qPCR assay to detect low concentrations of male and female DNA in mixed samples. We also test experimental DNA samples using our transfer DNA protocol to differentiate primary and secondary DNA transfer. Male DNA was detected in the majority of known mixed samples, even in samples with 4× more female DNA—this result demonstrates the ability to detect low concentrations of male DNA and the presence of secondary transfer DNA in our experimental design. Primary DNA transfer was detected in 100% of our experimental trials and secondary DNA transfer was detected in 37.5% of trials. Our innovative protocol mimics realistic case scenarios to establish rates of primary and secondary DNA transfer in an inexpensive and simplified manner.     

“YFlag” – A Single‐base Extension Primer Based Method for Gender Determination

Assigning the gender of a DNA contributor in forensic analysis is typically achieved using the amelogenin test. Occasionally, this test produces false‐positive results due to deletions occurring on the Y chromosome. Here, a four‐marker “YFlag” method is presented to infer gender using single‐base extension primers to flag the presence (or absence) of Y‐chromosome DNA within a sample to supplement forensic STR profiling. This method offers built‐in redundancy, with a single marker being sufficient to detect the presence of male DNA. In a study using 30 male and 30 female individuals, detection of male DNA was achieved with c. 0.03 ng of male DNA. All four markers were present in male/female mixture samples despite the presence of excessive female DNA. In summary, the YFlag system offers a method that is reproducible, specific, and sensitive, making it suitable for forensic use to detect male DNA.     

DYF155S1基因座的法医学应用研究

目的探讨Y染色体小卫星DYF155S1基因座在法医学中的应用价值。方法采用荧光MVR-PCR和Amp-FLP技术。结果同一个体不同组织所获得的分型结果一致;本方法能作出正确分型的最低基因组DNA量为0.3 ng;在女性成份为男性成份100倍的混合DNA样本(总DNA量多少为110 ng)中能正确检出DYF155S1基因型;调查130个父系家庭的男性成员样品(减数分裂336次),总共发现DYF155S1基因座有3次突变,突变率为0.9%。对强奸案混合斑的分析,可直接检出DYF155S1基因型。但是,本方法只能区分哺乳类雄性动物与非哺乳类动物。结论采用荧光MVR-PCR和Amp-FLP方法分析Y染色体小卫星DYF155S1基因座,方法可靠,灵敏度高,对混合斑和微量检材中男性DNA的分析具有较高的应用价值。