6FAM-HEX-TMR-ROX四色荧光分析体系的构建

目的为在310基因分析仪上进行6FAM-HEX-TMR-ROX四色荧光STR复合扩增分型建立基础条件。方法以pGEM载体作为靶DNA,设计引物扩增获得500~80bp的13个长度不等DNA片段,以这些片段纯化物作为模板DNA,分别扩增获得6FAM、HEX、TMR和ROX标记的M atrix标准物,用4种M atrix标准物在310基因分析仪上构建可用于6FAM-HEX-TMR-ROX四色荧光分析的M atrix文件。结果扩增所得的13个非标记片段,长度分别为80、100、120、140、160、180、200、220、260、300、340、400、500bp,在非变性聚丙烯酰胺凝胶连续电泳-银染凝胶上均表现为单条带,6FAM、HEX、TMR、ROX分别标记的片段通过310基因分析仪检测均为单峰。混合ROX标记的80、120、180、220、260、300bp的6个片段获得的内标,显示出良好的线性关系。结论建立了有效的6FAM-HEX-TMR-ROX四色荧光分析M atrix,为进行多个STR基因座荧光标记复合扩增检测奠定了基础。     

线粒体16srRNA和ND4基因在种属鉴定中的应用研究

目的构建一种用于种属鉴定的线粒体DNA(m tDNA)16 srRNA和ND4基因荧光标记复合扩增检测体系。方法利用引物设计软件(Prim er 5)对两个m tDNA序列ND4基因和16 srRNA基因设计两对引物,每对引物中的一条在5’端标记荧光素(6-FAM)。按传统复合扩增技术建立复合扩增体系,用AB I PR ISM 310基因分析仪对产物进行分析。结果人类DNA扩增产物出现两个峰,片段大小分别为110bp的人类特异片段和149bp的人与动物共有片段,而动物DNA扩增产物出现一个峰,片段大小为149bp。对30个实验室存放5~15年的陈旧人血痕也能明确判断其种属来源。结论该体系可以明确区分人源性生物检材与其它常见动物样本,对实验室长期存放的陈旧检材也具有较好的检测能力。     

2个miniSTR等位基因分型标准物的制备

目的采用分子克隆技术制备miniSTR D3S4529和D12ATA63基因座等位基因分型标准物,并评价其应用价值。方法用荧光引物对835份无关个体血卡样本进行扩增并分型检测,筛选2个基因座的等位基因片段,用分子克隆方法制备等位基因分型标准物,并对中国汉族群体进行遗传学调查。结果根据筛选出的等位基因片段制备出分型标准物,各等位基因峰形尖锐,无双肩峰,峰高基本一致,荧光值在4 000 RFU左右。中国汉族人群D3S4529、D12ATA63基因座分别检出7个、11个等位基因,杂合度分别为0.752、0.723,多态信息含量均为0.71。结论通过分子克隆法制备的miniSTR D3S4529和D12ATA63等位基因分型标准物,在法医学研究中具有较高的应用价值。     

荧光标记短片段STR复合扩增系统的法医学应用研究

目的解决严重降解(低于300bp)DNA检验问题,提高降解DNA的检出率。方法重新设计引物,减小扩增产物片段长度,用荧光标记短片段STR复合扩增体系进行DNA检验,扩增结果与用Identifiler试剂盒扩增出的结果进行比较。结果用荧光标记短片段STR复合扩增系统对实际案件中的高度降解DNA检材进行检验,可以获得满意分型。结论该系统可用于严重降解DNA检材的检验工作。     

法医DNA标准物质备选细胞STR等位基因片段长度的定值

目的 研究建立法医DNA标准物质备选细胞基因组STR基因座等位基因片段长度标准定值的方法.方法 利用有机法提取HPF和HSSM细胞基因组DNA并进行STR复合扩增,将产物进行电泳检测.利用Gene Mapper软件分析电泳结果,记录STR基因座等位基因的数值,并对目前我国公安系统应用较为广泛的DNATyperTM15、IdentifilerTM两种试剂盒扩增产物进行相应的DNA片段长度(bp)统计以及定值.结果 HPF为男性个体细胞,HSSM为女性个体细胞.HPF和HSSM细胞DNATyperTM15系统等位基因片段长度范围分别为126.26±0.05～367.53±0.20bp和125.33±0.07～370.08±0.17bp,IdentifilerTM系统等位基因片段长度范围分别为117.22±0.04～340.02±0.08bp和117.21±0.03～323.86±0.09bp.结论 对STR基因座等位基因片段长度进行标准定值,可为法医DNA标准物质提供有效的溯源途径.     

线粒体DNA短片段复合扩增系统鉴别种属的研究

目的建立线粒体DNA短片段复合扩增体系用于种属鉴定的方法。方法提取人、牛、猪、羊、鸡的DNA,用所选的3对引物复合扩增细胞色素b基因（cyt b）片段、16srRNA基因片段和ND4基因片段,扩增产物经琼脂糖凝胶电泳检测。结果人DNA扩增产物在358bp、157bp和110bp处各出现一条带;动物DNA扩增产物均只有358bp一条带。结论线粒体DNA短片段复合扩增鉴别种属的方法可区分人源性生物检材和其它动物样本,可应用于法庭科学实践。     

D1S549基因座分型标准物的克隆制备方法及其群体遗传学研究

目的 采用分子克隆技术建立制备大量优质标准 STR基因座等位基因分型标准物的方法,解决长期困扰 STR分型上存在的准确性和标准化问题。方法 先用 PCR扩增出 D1S549基因座的 8个等位基因片段,将其插入 pUC重组质粒中,经 DNA测序分析证实插入片段的结构及大小,用国际标准将插入的等位基因片段进行命名,最后经转染、扩大培养,扩增及再鉴定后制备出标准的 D1S549等位基因分型标准物。结果应用此分型标准物,调查了成都地区汉族 ,甘肃回族及新疆维族群体 D1S549基因座的基因型分布频率。结论 表明该法制备的 STR基因座等位基因分型标准物适用于法医学鉴定实践, D1S549基因座是一个适合我国群体分析和法医学遗传分析的遗传标记。     

miniSTR荧光检测体系的建立及法医学应用

目的 建立检测片段均小于150bp的miniSTR荧光检测体系,提高对微量降解检材DNA的检测效能. 方法 应用Primer Premier 5软件设计、FastPCR 6.0筛选引物,组合成用四色荧光标记引物的miniSTR复合扩增体系.优化PCR检测条件和引物浓度,在3100-Avant仪上用POP4胶进行电泳检测.分型结果用DNA标准品9947A和007进行验证,并通过检测新鲜血样、疑难微量检材评估该体系的法医学应用效能.结果 建立的miniSTR荧光检测体系（D12A TA 63、D2S1776、D1GA TA 113、D4S2408、D17S974、D20S482、D3S3053、Ame logenin、D6S474、D9S1122）中各基因座的检测片段均小于150bp,各等位基因扩增均衡性良好,无非特异性扩增产物,等位基因频率分布符合Hardy-Weinberg平衡,累积个体识别率为0.999999983,三联体累积非父排除率为0.996 8.能成功检测腐败肌肉组织、低拷贝数DNA检材以及在40％甲醛溶液中固定12d的人体组织. 结论 miniSTR荧光检测体系可独立应用于降解DNA样本的个体识别鉴定或补充应用于亲权鉴定,提高对微量、降解检材DNA的检测能力.     

荧光标记3个miniSTR基因座复合扩增系统的建立

目的建立扩增片段<135bp,包括D5S818,D8S1179,D16S539 3个miniSTR基因座复合扩增系统。方法采用不同荧光染料标记引物,通过PCR扩增,利用ABI 3100遗传分析仪进行片段长度分析,对100份无关个体血样,10个家系样本以及30份高度降解检材进行检测。结果本系统DNA分型结果与AmpFLSTR Identifiler试剂盒完全一致,且灵敏度高于AmpFLSTR Identifiler试剂盒。结论本系统可以应用于个人识别和亲权鉴定,为降解DNA样本分型提供了新的方法。     

利用人类c-Ha-ras-1埃可基因顺序串联28-bp重复子显示DNA纹印图谱

<正> 利用人类c-Ha-ras-1基因中28bp顺序串联重复子作为一个探针研究DNA纹印。以质粒PET为载体制备这个探针。从日本人外周血样中获取高分子量DNA。用识别位点为4个碱基的限制性内切酶Hinf·I或Sau3AI消化上述DNA。我们观察了洗脱条件不强烈情况下的由     

复合扩增mtDNA-HVI和Cyt b片段鉴定混合血痕种属

目的探讨mtDNA-HVI和Cyt b片段复合扩增法鉴定人与动物混合血痕种属的应用价值。方法用chelex-100法从人、牛、猪、狗、兔、鱼、鸡和鼠血痕中提取DNA,复合扩增mtDNA-HVI片段和Cyt b片段,琼脂糖凝胶电泳检测。结果人类在mtDNA-HVI区和Cyt b区分别出现279bp和358bp各一条带,且279bp条带亮于358bp;动物均只有358bp一条带。人与7种动物血痕的检测灵敏度均为3.13ng。检测人与动物混合DNA,灵敏度仍为3.13ng,但358bp条带亮于279bp条带。结论当358bp带明显强于279bp带时,提示检材为人与动物的混合。     

考古样品中Amelogenin同源基因的提取和检测

目的利用人类性染色体Amelogenin同源基因在X、Y染色体上序列长度的差异,选择设计引物,对考古样本进行古DNA性别信息研究。方法采用苯酚/氯仿-二氧化硅-超滤离心方法提取东岭墓葬群殉人骨骼、牙齿古DNA,PCR扩增,非变性聚丙烯酰胺凝胶电泳(PAGE)分离和检测古DNA扩增片段。结果8个墓葬16个样品中有7个样品出现阳性扩增检测,目标基因片段清楚,男性为二条带(X、Y),女性为一条带(X),牙齿样品检测成功率优于骨骼样品。结论改进的苯酚/氯仿—二氧化硅—超滤分离法是较好的古DNA提取方法,具有降低PCR抑制剂、消耗成本低和提取成功率高等特点。基于人类X、Y染色体Amelogenin同源基因的古DNA性别分析方法可成为分子考古重要的技术方法。     

Reliability of SE33 typing by capillary electrophoresis

A ladder of 24 ACTBP2 (SE33) alleles was separated 175 times by denaturing capillary electrophoresis on an ABI Prism 310 Genetic Analyzer using polymer POP-4. The mean standard deviation of fragment size determination was 0.083 bp. Fragments in the whole allelic range of ACTBP2 could be typed with high precision and reproducibility if adjacent fragments differed by at least two nucleotides. The capacity of resolving 1 bp differences was tested by repeatedly running a ACTBP2*14.2/14.3/31.2/31.3 allelic mixture. The 14.2/14.3 fragment pair could be separated in 98%, the 31.2/31.3 fragment pair only in 65% of all runs. Reliable separation of this difficult fragment mixture could exclusively achieved by using POP-6.     

Establishing the rDNA IGS structure of Cannabis sativa

The rDNA intergenic spacer (IGS) structure of Cannabis sativa was established and can be used for classification and identification of this species. In this study, DNA fragments of rDNA IGS were amplified by PCR from Cannabis sativa plant extracts and a 1387 bp fragment was obtained. DNA sequence analysis revealed six different repeat motifs. In the middle of the IGS sequence, there were three sequence motifs, and the same three sections of DNA were then repeated with minor variation in sequence. The terminal region of the IGS was composed of another three different repeat units; multiple copies of these terminal repeat motifs were present in no discernible order. Within six repeat motifs, point variations were observed in five. The DNA sequence of the locus was compared with all the plant sequences registered in GenBank by the Fasta program of GCG software with the result that this DNA fragment was significantly different from any other DNA sequence recorded to date. The most similar sequence was that of Hops (Humulus lupulus), but with a similarity of only 88.9% over 579 bp. These specific and complex variations of IGS may be related to the species and geographic distributions.     

A LDR‐PCR Approach for Multiplex Polymorphisms Genotyping of Severely Degraded DNA with Fragment Sizes <100 bp*

Abstract: Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini‐short tandem repeat‐polymerase chain reaction (PCR) and mini‐sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small‐scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.     

RAPD和ISSR分子标记检测大麻的遗传多样性初探

目的利用随机扩增多态性DNA和简单序列重复区间扩增分子标记检测大麻遗传多样性,并探讨其在法医学中的应用价值。方法收集中国4省6个地区的100株大麻叶子样品,采用CTAB法提取基因组DNA,设计选择11个RAPD引物和13个ISSR引物,采用6%中性聚丙烯酰胺凝胶电泳-硝酸银染色法进行检测,根据出现的条带数目和片段大小等分析大麻的多样性。结果 11条RAPD引物扩增出的片段在200bp以上共52条,其中具有多态性的27条;ISSR引物扩增出126条,其中具有多态性的73条;多态性条带比率分别为51.9%和57.9%,其差异不具有统计学意义(P>0.05)。结论 RAPD和ISSR两种方法均可用于大麻遗传多样性分析,对检测毒品原植物的种类和来源地具有一定的应用前景。     

Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies

We explore different designs to estimate both nuclear and mitochondrial human DNA (mtDNA) content based on the detection of the 5' nuclease activity of the Taq DNA polymerase using fluorogenic probes and a real-time quantitative PCR detection system. Human mtDNA quantification was accomplished by monitoring the real-time progress of the PCR-amplification of two different fragment sizes (113 and 287 bp) within the hypervariable region I (HV1) of the mtDNA control region, using two fluorogenic probes to specifically determine the mtDNA copy of each fragment size category. This mtDNA real-time PCR design has been used to assess the mtDNA preservation (copy number and degradation state) of DNA samples retrieved from 500 to 1500 years old human remains that showed low copy number and highly degraded mtDNA. The quantification of nuclear DNA was achieved by real-time PCR of a segment of the X-Y homologous amelogenin (AMG) gene that allowed the simultaneous estimation of a Y-specific fragment (AMGY: 112 bp) and a X-specific fragment (AMGX: 106 bp) making possible not only haploid or diploid DNA quantitation but also sex determination. The AMG real-time PCR design has been used to quantify a set of 57 DNA samples from 4-5 years old forensic bone remains with improved sensitivity compared with the slot-blot hybridization method. The potential utility of this technology to improve the quality of some PCR-based forensic and ancient DNA studies (microsatellite typing and mtDNA sequencing) is discussed.