Comparative Analysis of the HV1 and HV2 Regions of Human Mitochondrial DNA by Denaturing High-Performance Liquid Chromatography

Abstract:  Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a sequencing-independent means of detecting the presence of sequence differences in pair-wise mixtures of nonconcordant amplicons of human mitochondrial DNA (mtDNA). A total of 920 pair-wise combinations of HV1 and HV2 mtDNA amplicons from 95 individuals were assayed by DHPLC for sequence concordance/nonconcordance. For the 72 combinations of amplicons from different individuals who shared identical DNA sequences, DHPLC assays consistently indicated sequence concordance between the samples. This was in 100% agreement with sequencing data. For the 849 combinations of amplicons which differed in sequence, DHPLC detected the presence of sequence nonconcordance in all but 13 assays to yield 98.5% concordance with sequencing. Thus, DHPLC can be used to detect a diversity of sequence differences (transitions, transversions, insertions, and deletions) in the mtDNA D-loop. Accordingly, DHPLC may have utility as a presumptive indicator of mtDNA sequence concordance samples, as a screen for heteroplasmy/situational mixtures, and as a means for the physical fractionation of the individual contributors to an mtDNA mixture prior to sequencing.     

Concordance Between the AmpFℓSTR® MiniFiler™ and AmpFℓSTR® Identifiler® PCR Amplification Kits in the Kuwaiti Population

Abstract:  The AmpFℓSTR^® MiniFiler™ polymerase chain reaction amplification kit, developed and supplied by Applied Biosystems, complements the AmpFℓSTR^® Identifiler^® polymerase chain reaction amplification kit (Applied Biosystems, Warrington, U.K.) by improving the success rate when profiling DNA that is degraded or contains inhibitors. Before applying the MiniFiler™ kit to casework, the profiles from 200 unrelated Kuwaitis were compared to Identifiler^® profiles. Concordance was observed for 99.875% (1598 of 1600) of the compared STR loci. The two discordant profiles displayed allelic dropout: one at the D13S317 locus due to nonamplification of allele 10 in the MiniFiler™ profile, and one at the D18S51 locus due to nonamplification of allele 18 in the Identifiler^® profile.     

Degeneration of Nuclei and Mitochondria in Human Hairs

Abstract:  It is generally accepted that nuclei degrade in developing hair shafts but the point at which such occurs has not been investigated. The fate of mitochondria in the keratinizing hair shaft has been less clear. This study uses transmission electron microscopy to investigate when nuclei and mitochondria are no longer visible in the developing hair shaft. Serial sections were obtained from anagen head hairs absent follicles in order to determine the sequence of degradation of nuclei and mitochondria in the hair shaft by starting at the root bulb and proceeding toward the hair tip. It was demonstrated that nuclei and mitochondria become invisible in the keratinizing hair shaft at about the same time. This was found to occur fairly early in the process at the level of the hair shaft where the hair cuticle becomes permanent.     

Application of DNA Forensic Techniques for Identifying Poached Guanacos (Lama guanicoe) in Chilean Patagonia*

Abstract: Guanaco (Lama guanicoe) is a protected and widely distributed ungulate in South America. A poacher, after killing guanacos in Valle Chacabuco, Chilean Patagonia, transported and stored the meat. Samples were retrieved by local police but the suspect argued that the meat was from a horse. Mitochondrial cytochrome b gene (774 pb), 15 loci microsatellites, and SRY gene were used to identify the species, number of animals and their population origin, and the sex of the animals, respectively. Analysis revealed that the samples came from a female (absence of SRY gene) Patagonian guanaco (assignment probability between 0.0075 and 0.0282), and clearly distinguishing it from sympatric ungulates (E‐value = 0). Based on the evidence obtained in the field in addition to forensic data, the suspect was convicted of poaching and illegally carrying fire arms. This is the first report of molecular tools being used in forensic investigations of Chilean wildlife indicating its promising future application in guanaco management and conservation.     

Validation of the AMPFℓSTR® MiniFilerTM PCR Amplification Kit for Use in Forensic Casework*

Abstract: The AmpF?STR^® MiniFiler^TM PCR Amplification Kit is designed to genotype degraded and/or inhibited DNA samples when the AmpF?STR^® Identifiler^TM PCR Amplification Kit is incapable of generating a complete genetic profile. Validation experiments, following the SWGDAM guidelines, were designed to evaluate the performance of MiniFiler. Data obtained demonstrated that MiniFiler, when used in conjunction with Identifiler, provided an increased ability to obtain genetic profiles from challenged samples. The optimum template range was found to be between 0.2 and 0.6 ng, with 0.3 ng yielding the best results. Full concordance was achieved between the MiniFiler kit and Identifiler kit except in a single case of a null allele at locus D21S11. Numerous instances of severe heterozygous peak imbalance (<50%) were observed in single source samples amplified within the optimum range of input DNA suggesting that caution be taken when attempting to deduce component genotypes in a mixture.     

Validation of the Short Amplicon Multiplex Q8 Including the German DNA Database Systems*

Abstract: We have developed a concept to enable the analyzing of degraded stains with limited DNA template quantity. Therefore we have constructed a short tandem repeat (STR) multiplex including the German DNA database systems (Q8). The amplicon lengths are smaller than 280 bp. For the validation of Q8 over 50 degraded samples were investigated. Amplifications were performed with “low copy number” PCR, the number of PCR cycles was increased to 33 and the reaction volume was decreased to 12.5 μL. Compared with the MPX2 and Nonaplex kit, the average success rate was increased using the Q8 kit by approximately 20% and 30%, respectively. The efficiency of a sensitive STR multiplex with reduced amplicon lengths was confirmed in comparing the success rates of Q8 for typing degraded samples and samples with limited amount of DNA template while partial profiles were observed with the majority of the samples using commercially available kits.     

法医物证DNA自动化检验技术体系的研究

目的建立自动化工作站同步提取不同种类涉案法医生物检材DNA的新方法。方法选用TECAN Freedom EVO100．4、75—2型自动化提取、加样工作站,采用磁珠法及Chelex-100法对各类涉案生物检材进行DNA提取、PCR扩增、毛细管电泳检测其STR分型,进行比较测试。在“全国公安机关DNA数据库应用系统”中建立并应用实验室信息管理系统（LIMS）模拟实施规范化DNA检案。结果1552份各类检材,采用工作站-磁珠法提取DNA效果最佳,STR检测成功率为95％,工作站-Chelex法为88％;二者分别与其手工提取法比较,成功率无明显差异。92个样本同期检测,自动化工作站较手工操作DNA检案时间可缩减1．25倍。结论工作站域珠法提取涉案检材DNA,可获得满意的STR分型结果。应用LIMS管控,可有效防控污染,明显提高检案效率及鉴定质量。     

人体死后肝细胞DNA含量与死亡时间关系的研究

目的研究人体死后肝脏细胞DNA含量变化与死亡时间的关系及影响因素。方法选取46例已知死亡时间的人体肝脏,根据离体肝脏所处的环境温度分为12—19％（A组）和20—27％（B组）两组,每组23例。在死后24～72h内每隔4h穿刺取肝组织1次,制成细胞悬液,经RNA酶消化,PI染色后,用流式细胞仪测定被检测细胞中含不完整DNA的细胞数所占百分比,所得数据经Exp032V1．2软件计算N值。结果死后24～72h肝细胞N平均值,A组从10．91％增至49．72％,B组从16．22％增至69．63％。两组N平均值随死亡时间的延长均逐渐增高,与死亡时间有相关性,A组r值为：0．598,B组r值为0．77357。并且建立了不同环境温度对应的回归方程。结论在不同环境温度下,死后24～72h内人体肝脏细胞DNA降解均随死亡时间的延长和环境温度的升高而逐渐加快,相关数据可望为死亡时间推断提供一定参考依据。     

刑事案件DNA检验采样与鉴定立法现状

人类DNA遗传特征多态性使DNA技术成为各国警方侦办案件的重要技术手段,但DNA检验鉴定采样合法性和结论可采性须由立法规定。众多国家和地区针对DNA采样和鉴定结论应用制定了相关法律法规。我国相关法律细则尚处空白,因此,应尽快制定我国DNA鉴定采样与应用法规,规范DNA鉴定样本采集与证据采信。     

美国“DNA行动计划”对我国的启示

DNA检测技术问世已近30年,在侦查破案和维护司法公正方面发挥着越来越重要的作用。随着DNA检测范围的不断扩大、案件数量的急剧增长,人们发现了许多制约DNA充分发挥证据效力的问题。为了更有效地利用DNA证据,最大限度发挥DNA技术打击犯罪和保护无辜的科技威力,从2004年起,美国开始实施为期5年、耗资10亿美元的“总统DNA行动计划”。这一划时代的DNA国家战略规划成为美国司法部门应用和发展DNA技术一个全面的行动纲领,同时．该项目的实施进展情况也为我国当前和今后更好地应用DNA技术提供了许多宝贵的启示和借鉴。