用PCR技术对腐败尸体的软骨进行性别鉴定

本文应用PCR技术[1]对腐败抵抗力强的软骨进行了性别鉴定，结果满意。现报告如下。材料和方法检村：取自现场提取的腐败尸体肋软骨组织，气管软骨组织。己知男性的外周血作为对照。试剂：引物Y3、Y4（复旦大学遗传研究所设计）。TaqDNA聚合酶（美国PE公司）。DNA的提取：软骨组织的脱钙在周先略的方法[2]上进行了修改。首先将软骨组织块用30％双氧水浸泡24h（室温），取出用蒸馏水反复漂洗。在软骨块上钻孔后，放入氯仿：甲醇（3：1）浸泡1h，丙酮浸1h。用蒸馏水反复漂洗，切成薄片放入5％EDTA内24h，换液一次，取出用蒸馏水反复漂…     

法医学中的性别鉴定

对用形态学、细胞学、性激素检测、 DNA重组技术和聚合酶链式反应（ PCR）等方法进行性别鉴定进行了概述,显示骨骼的性别鉴定以形态学方法为好;对能获得足够量基因组 DNA的人体组织,最好用 PCR检测牙釉基因。而检测性染色质因阳性率较低,检测性激素仅适用于血痕检材, DNA分子杂交技术需要复杂的仪器及放射性同位素标记的探针,这些方法目前已较少应用。     

耻骨联合面推断年龄显著偏差1例

1案例在某工地旁发现一具白骨化无名女尸。提取耻骨联合经水煮法后，联合面见嵴残痕，背侧缘腹侧斜面形成，据此推断年龄25岁左右。经侦查人员查找尸源，根据死者衣物及义齿特征认定了死者，实际年龄为36岁。2讨论此案例之所以出现年龄推断与实际差距大，笔者认为，可能受以下因素影响：⑴性别因素，女性耻骨联合面的形态变化有的较男性早3～4年，有的较男性晚7～8年，故在根据耻骨联合面鉴定年龄时应考虑性别因素，再鉴定骨龄。⑵个体各躯干骨之间的发育存在时间上的差异。本例提取的死者颅骨经水煮法后，基底缝消失，愈合处见浅沟嵴，提示…     

损伤组织出血细胞自溶的特殊染色法

在法医病理学鉴定工作中,常遇到死亡时间较长的组织创伤案件,由于组织中红细胞自溶碎裂后与胶原纤维等混杂,用常规HE组织切片染色难以判别系出血、生前伤或死后伤。本研究借用区别胶原纤维、红     

机体损伤伴发癔病的法医临床学鉴定(附14例分析)

癔病作为最严重的神经症，临床表现依其类型的不同，分别在精神与躯体方面出现各种各样的症状。机体损伤伴发疫病，在法医临床检案中经常遇到。作者近几年检验鉴定的3000例案件中，遇有14例，占鉴定人数的4．7％，现报道如下：资料分析瘟病，在法医临床鉴定时，必须符合；（1）损伤刺激后病态发作；（2）排除器质性神经损害病变；（3）经临床暗示性治疗症状有好转或当获益性赔偿后症状自行好转、停止发作及消失。以下对损伤诱发症病的关联性问题进行分析。1．性别、年龄与文化程度本文机体受到伤害伴症病发作的14例中，男性1例，女性13例，…     

体外DNA扩增技术鉴定血痕性别三例报告

本文报道用体外DNA扩增技术对3例刑事案中的人血痕标本进行性别鉴定。其方法是从血痕标本中微量抽提DNA,用蛋白酶K进行消化。然后用两组引物Y1.1与Y1.2和Alu9.1与Alu9.2及国产FD耐热DNA聚合酶进行聚合酶链反(PCR)扩增DNA,电泳分析Y及Alu重复序列,从而判断血痕的性别。     

分析扩增DNA序列测定人发的性别

最常用的DNA分析方法是测DNA的限制性片段长度多态性(restriction fragment length polymorphism, RFLP).此法需要微克量的未降解大分子DNA,一般难以从案例检材中获得.聚合酶链反应(polymerase chain reaction, PCR)能使DNA的特异区域扩增,供序列分析,鉴定生物性检材的性别.酶促扩增法可使     

家兔心肌自溶蛋白质降解与死亡时间的关系

本文对家兔心肌自溶时的蛋白质水解和氨基酸代谢进行了研究。心肌组织在37℃湿盒中自溶15～480min,用离子交换色谱法测定游离氨基酸以及组织氨含量。自溶时大多数组织游离氨基酸含量上升,丙氨酸、赖氨酸和组氨酸含量在自溶15min即有明显增加,而谷氨酸含量呈下降趋势,同时组织氨含量上升。结果提示:心肌自溶早期蛋白质降解加速。上述结果有助于死亡时间的推断。     

兔死后肝细胞自溶的超微结构观察

用透射电镜观察兔死后肝细胞的超微结构变化,发现：在0．5h即有轻微的自溶改变,表现为细胞核中异染色质异常凝集和滑面内质同扩张;在1h自溶变化已趋明显,且随死后时间延长,在2h、3h、4h、5h该变化越来越广泛而显著,可见线粒体肿胀,嵴断裂、消失,基质内絮状致密体（Flocculentdensehadies,FDB）形成,粗面内质网及核周池扩张等改变。肝细胞自溶的超微结构变化与死后时间相关,这对推断早期死亡时间有应用价值。     

皮肤电流斑“极化现象”冷藏半年不消失

某女，32岁、因家庭失和被其丈夫用木棒打击全身多处软组织，致肺脏大面积脂肪栓塞而死亡。尸检时，发现死者双小腿各有一处小创口，当时未取材镜检。后有人怀疑该二创口系电击所致。重新检验时，尸体已在冰柜内保有近半年，各部位已出现了不同程度的自港与腐败。经过创四周围多部位取材。表皮细胞核尚未消失，未见有“极化现象”存在。有人怀疑“极化现象”因死后自溶而消失。之后我们收集了三例电击致死，且有较明显电流斑的皮肤标本，分别放置于冷勤柜中1个月、3个月和6个月，切片镜检，见三例的“极化现象”均未消失，相比于新鲜组织，…     

Persisting fetal microchimerism does not interfere with forensic Y-chromosome typing

Forensic Y-chromosome typing applies Y-chromosomal polymorphisms to the analysis of male/female mixed stains such as vaginal swabs in rape cases. The sensitivity of this approach exceeds that of cytological techniques combined with autosomal DNA typing. Y-chromosome typing is based on the assumption that Y-chromosomal DNA found in tissue or secretions of women must originate from a male individual, usually the perpetrator. Nevertheless, it was shown recently that fetal cells can migrate into the female body during pregnancy and can persist for decades ("persisting fetal microchimerism"). The body of a woman after a pregnancy with a male embryo can thus display a small fraction of fetal cells with Y-chromosomes. Using high sensitivity PCR protocols (reamplification with nested primers and up to 60 PCR cycles) fetal cells were previously identified in a number of maternal tissues including skin, blood, muscle and solid organs. It is, however, not clear at present, whether these cells can occur in vaginal secretions, and whether they are capable of producing false positive results in forensic Y-chromosome typing. To evaluate these questions, 66 blood samples of women with at least one son and nine vaginal swabs of women without sexual intercourse in the last 2 weeks were amplified for a stretch of the SRY gene. Eight thyroid gland tissues with already established male fetal microchimerism were used as positive control samples. Blood samples of 10 young girls without history of pregnancy were used as negative controls. Using a PCR with 10 ng of extracted DNA and 30 PCR cycles ("routine sensitivity assay") none of the samples yielded positive results. However, in a PCR with 200 ng of extracted DNA and 45 PCR cycles ("high sensibility assay"), 14% of the blood samples of mothers and 33% of the vaginal swabs amplified for SRY. Our results thus show that increasing the sensitivity of the PCR method and the amount of template DNA produce positive results while protocols used for routine Y-chromosomal typing with small amounts of DNA (approximately 10 ng of DNA) and with a limited number of PCR cycles (approximately 30) can clearly eliminate this peril.     

Differences in tissue distribution of HV2 length heteroplasmy in mitochondrial DNA between mothers and children

Sequence analysis of HV2 in mitochondrial DNA has been performed as a tool for forensic identification, in addition to that of HV1. HV2 contains length heteroplasmy, which shows high variability within an individual or in maternal relatives. In this study, we used cloning analysis and PCR direct sequencing to compare, between mothers and their children, HV2 length heteroplasmic profiles in different tissues. For two mother-child pairs, different types of variant distribution were observed by cloning analysis. In pair 1, length heteroplasmic patterns in most tissues were similar (predominantly 9 and 10Cs variants), but different length heteroplasmic levels, with shifts in predominant genotype, were observed for some hairs in both mother and child. In pair 2, genotype distribution was similar for all tissues, with a predominant 8Cs genotype, but varying in the proportion of minor component. The proportion of one minor length variant (9Cs) in blood from the child was significantly higher than that from the mother, but the proportions of minor components (7 and/or 9Cs) in other tissue samples decreased from mother to child. Moreover, we could confirm that sequence types of PCR products were reflected by the distribution of length variants, which were observed especially in high proportion, in cloning analysis. Our results reveal variable changes in length heteroplasmic level in various tissues between generations. Variability between tissues, especially among hairs, within an individual would result in complicated differences in genotype distribution between maternal generations, and correlate with longer length of Cs for predominant variants.     

Quantification of mitochondrial DNA in human blood cells using an automated detection system

The 4977 bp deletion of mitochondrial DNA (mtDNA) accumulates in postmitotic tissues with advancing age. The purpose of our study was to detect and quantify these deletion even in blood cells with a high turnover activity. Whole venous blood, isolated human platelets and peripheral blood mononuclear cells (PBMCs) were collected from 10 unrelated donors aged 20-71 years and total DNA was extracted. PCR was performed for total and mutated mtDNA using two different primer pairs and two fluorogenic probes labeled with the fluorescent dyes FAM and VIC. Specific PCR products were generated, detected and quantified in a real-time PCR. The amplification products of total and deleted mtDNA could be detected in each sample and did not exhibit any differences in the amount of the deleted mtDNA in whole blood, human platelets or PBMCs. Our data did not show any accumulation of the 4977 bp deletion with increasing age as it was observed for several other tissues.     

The polymerase chain reaction and post-mortem forensic identity testing: application of amplified D1S80 and HLA-DQ alpha loci to the identification of fire victims.

The application of DNA typing methods after amplification by the polymerase chain reaction (PCR) of DNA derived from body tissues from charred fire victims was investigated. A total of 26 different tissue specimens from ten extensively burned individuals were analyzed. The samples included femoral muscle, psoas muscle, bone marrow and blood. The post-mortem period varied from 38 to 183 h. After amplifying the DNA by PCR from the various tissues, the D1S80 locus was analyzed with a high resolution polyacrylamide gel electrophoresis technique followed by silver staining and the alleles of the HLA-DQ alpha locus were detected by using a reverse dot blot format. All samples could be typed for both loci and the genotypes were consistent in the various tissues from each individual. A parentage test was performed in two cases and Mendelian inheritance of the alleles for both loci was observed.     

Optimal storage conditions for highly dilute DNA samples: a role for trehalose as a preserving agent

DNA extraction from trace samples or noninvasively collected samples often results in the recovery of low concentration solutions of DNA that are prone to DNA degradation or other loss. Because of the difficulty in obtaining such samples, and their potentially high value in wildlife and forensic studies, it is critical that optimal methods are employed for their long-term storage. We assessed the amplification yield of samples kept under different storage conditions with the addition of potential preserving agents. We stored dilutions of known concentration human placental DNA, and gorilla fecal DNA, under four conditions (+4 degrees C, -20 degrees C, -80 degrees C, dry at room temperature), and with three additives (Tris EDTA (TE) buffer, Hind III digested Lambda DNA, trehalose). The effectiveness of the treatment methods was tested at regular intervals using qPCR to assess the quantity of amplifiable DNA, and a PCR assay of a larger 757 bp fragment to evaluate the quality of that remaining DNA. The highest quantity of DNA remained in samples stored at -80 degrees C, regardless of storage additives, and those dried at room temperature in the presence of trehalose. Surprisingly, DNA quality was best preserved in the presence of trehalose, either dried or at -80 degrees C; significant quality loss occurred with -20 degrees C and +4 degrees C storage.     

Quantitative analysis of amplifiable DNA in tissues exposed to various environments using competitive PCR assays

Competitive PCR assays were established for the mitochondrial DNA hypervariable region I and the human amelogenin locus. Using these assays, the copy numbers of DNA participating in PCR (amplifiable DNA) were quantified in tissues exposed to different environments. Human ribs, skin and nails were left in three exposure conditions (in the open air, in soil and in water). The amounts of amplifiable DNA in these tissues were quantified during a time period of up to two months. The amount of amplifiable DNA was well preserved in hard tissues (ribs and nails) regardless of the exposure conditions, whereas the soft tissues immersed in water showed a rapid decrease in amplifiable DNA. Strong PCR inhibition was observed in the DNA extracts obtained from buried bones. This phenomenon was clearly identified from an amplification failure of the internal standards in the competitive PCR. A preliminary examination to identify the PCR inhibitor suggested that the soil itself contributed to the inhibition. In addition, the amounts of amplifiable DNA in case samples were also investigated.     

福尔马林固定石蜡包埋组织3种DNA提取方法比较

目的探讨经福尔马林固定1d石蜡包埋组织(FFPET)提取DNA的简易有效方法。方法比较水浴加热、微波加热和二甲苯脱蜡的效果。组织脱蜡后分别采用Chelex-100+层析柱纯化法、DNA IQTM试剂盒磁珠提取法和Chelex-100+磁珠纯化法提取DNA;实时荧光定量PCR技术定量DNA;荧光标记毛细管电泳技术进行STR分型。结果二甲苯脱蜡的效果好于其他两种加热的脱蜡方法(P<0.05)。Chelex-100+层析柱纯化所获得的DNA量显著高于其他两种方法(P<0.05)。结论二甲苯脱蜡、Chelex-100+层析柱纯化法是一种简单、有效的FFPET处理方法。     

部分腐败尸体的亲权鉴定——附案例报告

部分腐败尸体的亲权鉴定在法医实践中常会遇见,过去的血型测定法已无能为力。PCR 技术给这类案件的鉴定带来了希望。本文报告用 PCR 技术对部分腐败的女性尸体和胎儿进行基因分型并对此案的亲权进行鉴定,取得了令人满意的结果。同时讨论了一些注意事项。作者认为 PCR 技术在鉴定此类案件中有特殊价值。     

Peculiarities of detection and evaluation of the persistence of tetraethyltiuram disulfide in the biological material

Optimal conditions for the identification and quantitative determination of tetraethyltiuram disulfide in "fresh" and putrefactive tissues of cadaverous liver are described for the purpose of TLC, HPLC, and IK-spectrophotometry following extraction of the compound of interest with ethyl acetate and its purification on a silicagel L column, 40/100 mcm. The persistence of tetraethyltiuram disulfide in the cadaverous tissues was evaluated. It was shown that the period during which tetraethyltiuram disulfide can be detected in the autopsied tissues decreases from 203 to 28 days with a rise in temperature from -15 degrees C to +36 degrees C.     

Postmortem production of ethanol in different tissues under controlled experimental conditions

The aim of this study was to follow the postmortem ethanol production phenomenon under controlled experimental conditions (temperature, time interval) in different tissues. Specimens of blood, liver, skeletal muscle and kidney were taken from 30 corpses and no chemical preservatives were used in the specimens collected. Ethanol concentrations were detected by gas chromatography. All specimens stored at -20 degrees C and 4 degrees C did not show any change in ethanol concentration in an eight-day time interval. At 20 degrees C and 30 degrees C, all tissues, except blood, showed statistically significant ethanol production over the time interval tested. However, blood sample kept at 30 degrees C, showed statistically significant increase in ethanol production on the 2nd and 4th day comparing to the controls. Thus, we can state that postmortem ethanol production occurs in different tissues, and is increased at higher temperatures and, in general, it is in accordance with the course of time.