SSCP法mtDNA分型的法医学应用

目的探讨SSCP法mtDNA分型在法医鉴定实践中的意义。方法PCR扩增mtDNAHV-I和HV-II的序列多态性片段,直接用SSCP技术分型。对70个湖北汉族真三联家系和140例随机个体进行检测,比较家系中mtDNA的单倍型SSCP图谱。统计分析SSCP法用于两个高变区在母系确定、个人识别中的意义和鉴别能力。结果在70个家系中,母亲和孩子的HV-I、HV-IISSCP带型完全相同;家系中父亲与母子的HV-I图谱不同占98.57%;HV-IISSCP图谱不同占97.13%。140例随机个体的HVI、HVII区分别检出21、16种单倍型,GD值分别为0.9556、0.9356。结论mtDNA-SSCP分型在嫌疑人筛查和母系亲缘关系推断中有实际应用价值。     

Mitochondrial DNA identification of game and harvested freshwater fish species

The use of DNA in forensics has grown rapidly for human applications along with the concomitant development of bioinformatics and demographic databases to help fully realize the potential of this molecular information. Similar techniques are also used routinely in many wildlife cases, such as species identification in food products, poaching and the illegal trade of endangered species. The use of molecular techniques in forensic cases related to wildlife and the development of associated databases has, however, mainly focused on large mammals with the exception of a few high-profile species. There is a need to develop similar databases for aquatic species for fisheries enforcement, given the large number of exploited and endangered fish species, the intensity of exploitation, and challenges in identifying species and their derived products. We sequenced a 500bp fragment of the mitochondrial cytochrome b gene from representative individuals from 26 harvested fish taxa from Ontario, Canada, focusing on species that support major commercial and recreational fisheries. Ontario provides a unique model system for the development of a fish species database, as the province contains an evolutionarily diverse array of freshwater fish families representing more than one third of all freshwater fish in Canada. Inter- and intraspecific sequence comparisons using phylogenetic analysis and a BLAST search algorithm provided rigorous statistical metrics for species identification. This methodology and these data will aid in fisheries enforcement, providing a tool to easily and accurately identify fish species in enforcement investigations that would have otherwise been difficult or impossible to pursue.     

用PCR—测序法对人类线粒体DNA多态区的研究

用PCR方法，对线粒体DNA多态区15997至三6401区域进行扩增，产物DNA经纯化处理局直接进行自动化序列测定，得到了准确的序列结果。将得到的中国人mtDNA多态区序列与国外已报道的相应序列进行对比，证实我们所测序列与白种人相应序列之间存在碱基差异。     

用ESI-TOF-MS分型技术检测线粒体DNA高变区碱基组成的多态性

目的 用ESI-TOF-MS分型技术检测线粒体DNA的D环高变区,通过碱基组成分析其多态性.方法 在PLEX-ID技术平台上,分别对mtDNA高变区1(HVⅠ,15924-16428nt)和mtDNA高变区Ⅱ(HVⅡ,31-576 nt)进行碱基组成分析,考察mtDNA在华东汉族人群的多态性,并将该技术应用于一例特殊的亲子鉴定案件.结果 用ESI-TOF-MS分型技术检测线粒体DNA,在高变区Ⅰ的8个区段检见碱基组成的多态性,在mtDNA高变区Ⅱ的10个区段检见多态性.在所应用的亲子鉴定案例中,线粒体DNA标记成了常染色体STR基因座的重要补充,经高变区Ⅰ和高变区Ⅱ的碱基组成检测,最后排除了非母.结论 ESI-TOF-MS检测mtDNA的技术具有良好的应用前景,在一些特殊的案件中,该法可为最终获得可靠鉴定结论提供技术支撑.     

用PCR和ESI-TOF-MS技术检测线粒体DNA的异质性

目的用PCR和ESI-TOF-MS分型技术检测线粒体DNA（mtDNA）D环高变区,通过碱基组成分析mtDNA的异质性。方法从华东汉族群体选取12名无关个体,用PLEX-ID平台进行mtDNA分型。该平台使用12对引物,对mtDNA高变区1（HVⅠ,引物所跨区域为15893~16451）进行碱基组成分析;使用另外12对引物,对mtDNA高变区2（HVⅡ,引物所跨区域为5~603）进行碱基组成分析,考察mtDNA异质性频率。结果 mtDNA多态性区域的碱基组成信息反映出区段内有无异质性。在高变区Ⅰ的12个区段中,有3个区段表现出多聚C长度异质性：在mtDNA高变区Ⅱ（31~576）的12个区段中,有3个区段检见点异质性,另外5个区域检见Poly C长度异质性。结论群体调查表明,mtDNA的序列异质性多见于高变区Ⅱ的103~267区段,多聚C长度异质性多见于高变区Ⅰ的16124~16201、16157~16201、16182~16250区段和高变区Ⅱ的234~367、431~576区段。将mtDNA标记用于母系关系检验和（或）个体识别时,需要格外留意这些异质性信息,以免结论错误。     

Results of the 2003–2004 GEP-ISFG collaborative study on mitochondrial DNA: Focus on the mtDNA profile of a mixed semen-saliva stain

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003–2004. Five reference bloodstains from five donors (M1–M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1–M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1–M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.     

三个柔嫩艾美球虫虫株线粒体cox 1基因部分序列的比较分析

为研究柔嫩艾美球虫不同毒力株的线粒体细胞色素c氧化酶第1亚基(cox1)基因与球虫种群之间的遗传关系,应用聚合酶链反应扩增柔嫩艾美球虫3个不同毒力株的cox1序列,并与GenBank上登录的鸡柔嫩艾美球虫、巨型艾美球虫、毒害艾美球虫和堆形艾美球虫虫株的相应序列进行比对分析.结果显示,每个虫株都获得659 bp的cox1部分有效序列(pcox1).柔嫩艾美球虫不同虫株的pcox1序列完全相同,但与其他种的艾美球虫相应的pcox1序列有不同程度的差异.表明柔嫩艾美球虫的cox1序列可作为艾美球虫不同种虫株之间遗传变异研究的标记.     

粪便DNA提取及检验

目的　研究人类粪便DNA的提取和检验方法。方法　 8人份粪便样本 ,磁珠法提取DNA后 ,进行STR复合扩增和mtDNAHVI区测序分析。结果　用 2种方法提取的粪便DNA ,STR复合扩增检验均未获成功 ;方法1提取的粪便DNA有 6个样本、方法 2有 7个样本获得了清晰可读的mtDNAHVI区序列 ,并与唾液对照样本DNA的序列完全一致。结论　用本文建立的方法提取粪便DNA ,不适于STR分析 ,可通过mtDNA测序分析进行检验。     

人类线粒体DNA异质性研究进展及在法医学中的应用

线粒体DNA(mtDNA)异质性的存在使其在法医学应用变得复杂。本文对mtDNA异质性形成的可能原因、异质性的分布和遗传特点、异质性的筛查和定量方法、异质性对法医学的影响以及异质性的研究和展望等方面进行综述,探讨异质性在法医学上的应用价值。     

染色毛干mtDNA不同提取方法的研究

目的为染色毛发线粒体DNA选择最佳的提取方法。方法在5个染发个体中各取5根头发,采用Chelex-100法、有机法、磁珠法、H2O2结合Chelex-100法及Chelex-100结合M icrocon100法等5种不同的提取方法提取染色毛发m tDNA,利用2%琼脂糖凝胶电泳检测扩增产物。结果Chelex-100结合M icrocon100方法提取染色毛发扩增m tDNA效果较好,其余方法无扩增产物。毛发的不同部位对扩增结果无影响。结论染色毛发采用恰当的提取方法可以提取到m tDNA模板,为法医日常检案提供帮助。