A Grass Molecular Identification System for Forensic Botany: A Critical Evaluation of the Strengths and Limitations*

Abstract: Plant material is frequently encountered in criminal investigations but often overlooked as potential evidence. We designed a DNA‐based molecular identification system for 100 Australian grasses that consisted of a series of polymerase chain reaction assays that enabled the progressive identification of grasses to different taxonomic levels. The identification system was based on DNA sequence variation at four chloroplast and two mitochondrial loci. Seventeen informative indels and 68 single‐nucleotide polymorphisms were utilized as molecular markers for subfamily to species‐level identification. To identify an unknown sample to subfamily level required a minimum of four markers or nine markers for species identification. The accuracy of the system was confirmed by blind tests. We have demonstrated “proof of concept” of a molecular identification system for trace botanical samples. Our evaluation suggests that the adoption of a system that combines this approach with DNA sequencing could assist the morphological identification of grasses found as forensic evidence.     

中国武汉汉族群体STR D13S325基因座的多态性研究

应用PCR ,聚丙稀酰胺凝胶垂直电泳及银染技术对 2 16例中国汉族人群STRD13S32 5基因座进行了多态性研究 ,首次获得了中国武汉汉族人群基因频率分布资料。检出 11个等位基因 ,30个基因型 ,基因频率分布符合Hardy Weinberg平衡。DP值为 0 92 92 ,三联体PE值 0 6 137,二联体PE值为 0 4370 ,PIC值为 0 775 5。观察 2 0 0次减数分裂未发现突变基因。研究证实D13S32 5是一个高多态性STR基因座标记系统。     

用新设计引物调查武汉汉族Penta D和Penta E基因座遗传多态性

目的　研究PentaD和PentaE基因座分型引物设计 ,调查PentaD和PentaE基因座在武汉汉族人群中的遗传多态性。方法　用重新设计的PentaD和PentaE基因座分型引物 ,采用热启动PCR和PAGE技术对 2 81名武汉地区汉族无关个体进行分型调查 ,并将其分型结果与Promega公司的PowerPlexTM16系统荧光标记复合扩增试剂盒分型结果进行比较。结果　新设计引物扩增产物的片段大小范围分别为 15 3～ 198bp和 10 7～ 2 12bp ,其分型结果与PowerPlexTM16系统的分型结果完全一致 ,且用银染法检测的灵敏度显著提高 (由 0 5ng提高到 0 2ng)。PentaD和PentaE基因座在武汉汉族群体分别检出 10个和 2 1个等位基因 ,其基因型频率分布均符合Hardy Weinberg平衡。家系调查证实了其等位基因的传递符合孟德尔遗传规律。两基因座的个体识别能力 (DP)分别为 0 92 62、 0 9860 ,非父排除率 (PE)分别为 0 665 1、 0 83 2 5。结论　新设计引物用于PentaD和PentaE基因座的分型检测准确可靠 ,两基因座多态性程度高 ,在法医学个人识别和亲子鉴定中具有使用价值。     

Frequency assessment of SNPs for forensic identification in different populations

Allele frequencies for 16 previously described autosomal SNPs were tested in 1020 unrelated individuals originating from three different continents (Africa, Asia and Europe). The populations analyzed included Africans from Benin Gulf (180), Asians from Mongolia (160) and Europeans from Italy (680).     

短串联重复位点ACTBP2(SE33)的扩增片段长度多态性研究

应用变性聚丙烯酸胺凝胶电泳（dn－PAGE）结合银染色技术对短串联重复（STR）位点ACTBP2（SE33）的扩增片段长度多态性（Amp－FLPs）进行了研究。在210名无关中国个体中观察到了25个等位基因，等位基因频率分布在0．007～0．093之间。基因型的分布符合Hardy－Weinberg定律，个体识别能力（DP）值为0．99，杂合度（H）为98．7％。七个家系分析的结果表明，该位点的遗传符合孟德尔遗传法则，未观察到变异。对几种常见的法医物证检材的分析表明，该分型系统对DNA降解放为严重的检村适用性强，而且灵敏度高（0．5ng），适合于法医学实际应用。     

用地高辛配基标记的pAW101探针杂交分析DNA多态性及其应用

将NaCl盐析法抽提人基因组DNA与地高辛配基标记DNA探针的方法相结合，检测了15个家系子代与亲代DNApAW101－EcoRI限制性片段的遗传关系。结合所测同工酶（EsD．GLOI．pGM；．Acp）的基因频率、按Essen－moller氏公式计算，父权概率均达到99．73％以上。本法经济、有效、实用、易于在国内普遍实验室开展。     

短串联重复序列D3S1359基因座的多态性

应用PCR、聚丙烯酰胺凝胶垂直电泳及银染技术对STRD3S1359基因座进行多态性调查,首次获得中国汉族人群的基因频率分布数据。检出19个等位基因,59个基因型,基因型频率分布符合Hardy－Weinberg平衡。观察352次减数分裂未发现突变基因,DP值为0．9380,三联体PE值0．6311,二联体PE值0．4568,PIC值0．7824。证实D3S1359是一个高信息含量的STR标记系统。     

中草药复合添加剂对虹鳟鱼增重及肝功能和结构的影响

为观察中草药复合添加剂富康163对虹鳟鱼生长性能、肝功能和结构的影响,选择18月龄的虹鳟鱼400尾,随机将其分为4组,Ⅰ组为对照组,Ⅱ、Ⅲ、Ⅳ分别在基础日粮中添加30、50和70g/kg富康163,试验期60d。结果显示:饲料中添加不同水平的富康163能提高增重率,并可不同程度地降低饲料系数;与对照组相比,试验组血清腺苷脱氨酶(ADA)活性、γ-谷氨酰转肽酶(γ-GT)活性、胆碱酯酶(CHE)活性、总胆红素(TBiL)浓度和肝指数无明显变化(P>0.05);Ⅰ、Ⅱ、Ⅲ组肝细胞界限清楚,核圆而大,核膜清晰,胞质丰富,而Ⅳ组有的肝细胞间分界不明显、胞内空泡化。结果表明,虹鳟鱼饲料中添加富康163能促进鱼体生长,提高营养物质利用率,对鱼肝的功能和结构无显著影响,建议虹鳟鱼饲料中富康163的添加剂量以50g/kg为宜。     

HCN4 Gene Variations in Sudden Unexplained Nocturnal Death Syndrome in the Southern Han Chinese Population,

Sudden unexplained nocturnal death syndrome (SUNDS) is widely considered to be related to hereditary fatal arrhythmias. Hyperpolarization‐activated cyclic nucleotide‐gated channel 4 (HCN4) channels are widely distributed in sinus myocytes and play a profound role in generating pacemaker electro‐activity in cardiomyocytes. In the present study, the potential correlation between HCN4 gene variations and the occurrence of SUNDS was investigated. Genomic DNA was extracted from blood samples of both 119 unrelated SUNDS patients and 184 healthy individuals and screened for candidate HCN4 gene variants. One missense heterozygous variant c.1578C>T (Ala195Val) and four synonymous heterozygous variants c.1552C>T, c.2833C>T, c.3823C>T, and c.4189C>A were discovered in the SUNDS cases. The missense variant c.1578C>T (Ala195Val) was absent in 163 recruited controls and 105 persons of the Southern Han Chinese population, had in‐silico prediction indications as damaging, and was reported prevalent in sudden infant death, and is thus likely to be involved in SUNDS.     

Validating the consistency of cSNPs analysis results between DNA and RNA using SNaPshot method

The use of coding region single nucleotide polymorphisms (cSNPs) was recently proposed as a potential method for individual identification because it allows mRNA profiling and DNA typing to be performed concurrently. Nevertheless, availability of this approach still needs some further validation in different aspect. In this study, we have initially selected several SNP loci located in mRNA molecules that were confirmed to be highly expressed in blood. Both coding regions (CDRs) and untranslated regions (UTRs) were taken into consideration during the screening. Genomic DNA (gDNA) and total RNA from venous blood samples were isolated, followed by the synthesis of first-strand complementary DNA (cDNA) using purified RNA samples. Subsequently, the genotypes of these SNPs were respectively determined with gDNA and cDNA by using SNaPshot method. The PCR primers for cDNA were designed to span an intron in order to ensure that the amplification products were not due to the presence of potential DNA contamination. In summary, our study revealed a high consistency of cSNP analysis results between DNA and RNA on capillary electrophoresis platform, which highlighted the potential use of cSNP in forensic investigation.