Sex identification in fresh blood and dried bloodstains by a nonisotopic deoxyribonucleic acid (DNA) analyzing technique

Deoxyribonucleic acid (DNA) specimens were prepared from blood or bloodstain extracts, and the content of a Y-chromosome specific DNA fragment was investigated by the Southern hybridization method using a nonisotopic staining technique. Thus obtained patterns of male DNA showed a clear band, whereas broad stains with some faint bands appeared on the patterns of DNA from both sexes. This method is expected to be a new powerful mean of forensic medical examination.     

Human blood stain identification and sex determination in dried blood stains using recombinant DNA techniques

DNA was extracted from human and non-primate dried blood stains. Human male and female specimens were readily distinguished by analysis with a Y-chromosome specific DNA probe. Human and non-primate blood stains were also readily differentiated using a repeat sequence (Alu) DNA probe. The potential power of recombinant DNA analysis in forensic science is discussed.     

根据Y-染色体特异DNA序列鉴定牙齿性别

人类牙齿的性别鉴定,是法医学个体识别中的一个重要部分。作者们应用限制性内切酶 HaeⅢ、Sau3A 水解60颗牙髓 DNA,琼脂糖凝胶电泳分离酶解片段,在紫外灯下直接观察。根据 Y-染色体特异 DNA 序列,3.4kb 长片段的有无,判断牙齿的性别。为牙齿性别的法医学鉴定提供了一种简单、准确的新方法。     

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。     

Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science

A genetic locus (D1S58, defined by DNA probe pMCT118) that contains a variable number of tandem repeats (VNTR) has been successfully amplified from a very small amount of genomic deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR). The DNA sequence of the locus was determined and was found to consist of a 16-base consensus sequence and flanking sequences. Oligonucleotide primers complementary to the flanking sequences were synthesized to serve as primers for amplification of MCT118 by the PCR method. Human genomic DNA isolated from blood (2 ng from each sample) was successfully amplified at the MCT118 locus, and polymorphic bands were detectable by ethidium bromide staining after electrophoresis on polyacrylamide gels. Determination of genotypes at this VNTR locus can now be routinely achieved within 24 h, without the need for Southern blots or radioactive materials. Furthermore, the small size (387 to 723 base pairs) of the DNA fragments produced in the PCR amplification permits good resolution of individual alleles that differ by only one repeat unit. The precise specification of the number of tandem repeats present in each allelic fragment is reproducible from one analysis to another.     

非同位素标记DNA探针初步研究

本文报导了将辣根过氧化物酶直接标记在单链 DNA 探针上,制备出非同位素的 DNA 探针,采用化学光增强法检测杂交结果,所得图谱清晰,容易判读。所制备出的 DNA 探针可用于人类性别鉴定和个体识别,为 DNA 检验技术推广应用开辟了新的领域。     

A study of sex identification of trace, dried bloodstains using a Y-chromosome-specific deoxyribonucleic acid (DNA) probe

A new method is discussed which examines trace, dried bloodstains by gel in situ hybridization using a Y-chromosome-specific deoxyribonucleic acid (DNA) probe to determine the sex of the bloodstain for forensic medicine application. The complete DNA is transferred directly by electrophoresis onto the gel intact, bypassing the possibilities of impurities contaminating the sample and of DNA degradation. The method has proven accurate for small (2.5-mm-diameter) samples aged up to eight years and is quick, simple, and easily read.     

Development of a human-specific real-time PCR assay for the simultaneous quantitation of total genomic and male DNA

A duplex real-time quantitative PCR assay was developed for forensic DNA analysis, which provides simultaneous quantitation of total genomic human DNA and human male DNA. The assay utilizes two spectrally resolved fluorogenic probes in a 5' nuclease (TaqMantrade mark) assay. Within the range of organisms empirically tested and based upon theoretical specificity using National Center for Biotechnology Information GenBank sequences, primer and probe sequences were shown to be human specific, and the Y-chromosome probe, male-specific. A mixture-challenge study resulted in accurate quantitation of 25 pg male DNA in a mixture of up to 1:5000 (male:female DNA). Additional experimental results include comparisons with the slot blot method and commercial real-time PCR kits. The assay developed addresses the shortcomings of the traditional slot blot method as well as the commercial real-time PCR kits. This method is shown to be specific, relatively simple, rapid, has low limits of detection, and consumes limited sample in addition to reporting both the male and total genomic DNA concentrations present.     

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。     

D17Z1探针点杂交DNA定量研究

本研究化学合成了高等灵长类特异性α卫星寡核苷酸片段(D17Z1),经辣根过氧化物酶标记、分子杂交、化学发光法检测,建立了高等灵长类特异性DNA精确定量的方法.制备了DNA浓度梯度标准对照.对人类DNA,猴、猪、牛、羊、鸡、兔、鱼、小鼠等常见动物DNA及JMl09大肠杆菌、入DNA、φ174DNA等微生物DNA进行了定量分析.结果表明,应用该方法对人类DNA定量不受非高等灵长类动物DNA与微生物DNA的影响,可实现组分定量;灵敏度测试,可对0.12ng的人类DNA进行定量,适用于法医DNA检验定量分析.     

人DNA指纹检测的初步研究

根据随机探针检测DNA限制片段长度多态性的原理和人与鼠的髓鞘硷性蛋白(MBP)基因cDNA有90％以上同源序列的事实,我们选用rMBP-cDNA 3'端非表达区高度重复顺序的0.81kb片段作探针,检测用HaeⅢ酶解的人DNA限制性片段结果可以分解出22条谱带,受检的30例无血缘关系的个体之间,没有两个人的谱带是完全相同的,显示出此方法的高度特异性。本文还比较了若干DNA片段作探针和几种限制性内切酶检测人DNA指纹的结果。     

Sex determination of blood stains with a recombinant DNA probe: comparison with radioactive and non-radioactive labeling methods

A recombinant DNA probe hybridizing specifically to human repeat DNA sequence (pHY10) of which about 3000 copies are present on the Y chromosome was used for sex determination of degraded DNA samples of blood stains. Human blood stains of male and female origin were readily differentiated with the pHY10 DNA probe. This radioactive technique enabled reliable and sensitive sex determination from blood or dried blood stains greater than 20 years old. Less than 1 microliter of blood or 1 piece of 0.5 cm length thread of blood stain from cotton fabric was sufficient for the test using dot blot hybridization. Compared with the radioactive labeling method, the photobiotin labeling method showed one thirtieth to one fiftieth lower sensitivity and presented some problems which are expected to be resolvable.     

不同方式标记非放射性探针原位杂交效果评价

目的比较不同方式标记非放射性探针的原位杂交效果。方法通过两种方法标记地高辛随机引物DNA探针和反意RNA探针,在大鼠损伤模型的皮肤组织上进行原位杂交。结果两种探针均可满足要求,以反意RNA探针在阳性染色深度和背景方面效果更佳。结论RNA探针在杂交效果方面优于DNA探针。     

Y-STR polymorphism in Central Anatolian Region of Turkey

Eight Y-chromosome specific STR (Y-STR) loci including DYS19, DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393 were investigated in a group of males from Central Anatolian Region of Turkey. Healthy 59 males living in this region for at least three generations were included in the study. PCR analysis was carried out with Y-STR specific primers on genomic DNA obtained from peripheral blood samples and size determination of PCR products was performed by silver staining following 6% polyacrylamide gel electrophoresis (PAGE). DYS388 was found to be the locus with lowest diversity (D) whereas DYS389II was the locus with highest diversity. The current study presented a framework of variation for the eight Y-STR loci in Central Anatolian population.     

Detection of sequence variation in the HVII region of the human mitochondrial genome in 689 individuals using immobilized sequence-specific oligonucleotide probes

We have developed a rapid, immobilized probe-based assay for the detection of sequence variation in the hypervariable segment II (HVII) of the mitochondrial DNA (mtDNA) control region. Using a panel of 17 sequence-specific oligonucleotide (SSO) probes immobilized on nylon membrane strips, we typed 689 individuals from four population groups. The genetic diversity value for each population was calculated from the frequency data, and the frequencies of distinct "mitotypes" in each group were determined. We performed DNA sequence analysis of 129 samples to characterize the sequences associated with "blanks" (absence of probe signals) and weak probe signals. Out of 689 samples, we observed five heteroplasmic samples (excluding the variable C-stretch beginning at position 303) using the immobilized SSO probe panel. The SSO probe strips were used for the analysis of shed hairs and bloodstains from several criminal cases in Sweden, one of which is described here. We conclude that this mtDNA typing system is useful for human identification and significantly decreases casework turnaround time.     

Evaluation of two DNA/RNA co-extraction methods for body fluid identification in forensics

Body fluid identification has become a field of interest in forensic casework as it can add value to particular investigative scenarios. Identifying the source of the biological material is not always an upfront task using conventional methods; therefore, profiling of specific mRNA markers can provide the answer. The implementation of RNA based analyses in forensic casework must focus on the quality and sensitivity of methods, starting with nucleic acid extraction, and without loss of DNA for STR profiling. In this work, two methods for DNA and RNA co-extraction were tested and compared: a commercial kit that uses a spin, mini column methodology, and a quick, simple nucleic acid isopropanol precipitation based protocol. Both methods simultaneously extract DNA and RNA, crucial for forensic casework and were tested in semen samples. Nucleic acid quantifications as well as purity assessment ratios (OD₂₆₀/OD₂₃₀ and OD₂₆₀/OD₂₈₀) were obtained by both methods to infer on the use of extracts in downstream applications such as PCR. The performance of the two tested protocols was further evaluated by analyzing two semen mRNA specific markers, PRM1 and SEMG1. When compared to the commercially developed kit, results suggest that the literature adapted protocol allowed more carryover of contaminants absorbing at 230 nm and 280 nm as the purity ratios were below the accepted standard ranges. Negative results for mRNA profiling supported the QC results obtained by spectrophotometry. On the hand, PRM1 and SEGM1 were positive in RNA samples extracted with the commercial kit.     

DNA甲基化标记法医学应用探讨

CpG的胞嘧啶在DNA复制后多数被甲基化,5-甲基胞嘧啶的分布是贮存表遗传信息的主要形式。近年来研究表明,DNA甲基化标记具有信息含量丰富、相对稳定、检测和结果处理方便、可与SNP联合分析等优点,是一种新的强有力的遗传分析工具。基因组的甲基化差异可用甲基化敏感性限制酶、重亚硫酸盐转化、Maxam-Gilbert裂解等技术来分析。人类基因组甲基化谱有时空特异性、亲源特异性、病理特异性等特征,在法医亲子鉴定、个人识别等方面有潜在应用价值。     

A collaborative study of the EDNAP group regarding Y-chromosome binary polymorphism analysis

A collaborative study was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the performance of Y-chromosome binary polymorphism analysis in different European laboratories. Four blood samples were sent to the laboratories, to be analysed for 11 Y-chromosome single nucleotide polymorphisms (SNPs): SRY-1532, M40, M35, M213, M9, 92R7, M17, P25, M18, M153 and M167. All the labs were also asked to submit a population study including these markers. All participating laboratories reported the same results, indicating the reproducibility and robustness of Y-chromosome SNP typing. A total of 535 samples from six different European populations were also analysed. In Galicia (NW Spain) and Belgium, the most frequent haplogroup was R1b*(xR1b1,R1b3df). Haplogroup F*(xK) is one of the most frequent in Austria and Denmark, while the lowest frequency appear in Belgium. Haplogroup frequencies found in this collaborative study were compared with previously published European Y-chromosome haplogroup data.