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。     

云南大麻DNA的提取及检测初步研究

目的探讨云南大麻DNA的提取及检测方法。方法运用改进的SDS微量法提取大麻总DNA,对所得DNA进行PCR检测。结果用所筛选到的大麻引物检测了云南大麻的DNA遗传标记特征,图谱清晰,结果稳定。结论本方法能有效提取并检测大麻DNA,为实现大麻的品种鉴定、遗传多态性研究及来源追踪提供有价值的科学依据。     

AFLP技术鉴别罂粟、虞美人和大麻种属差异的初步研究

目的探讨采用AFLP技术检测植物DNA,鉴别罂粟、虞美人和大麻植物种属间差异的方法。方法收集罂粟根、茎、叶、花、果以及虞美人和大麻叶检材,用AxyPrep DNA试剂盒提DNA,经EcoRI/MseI酶切,人工接头及PCR预扩增,用E-ACA/M-CAG、E-ACT/M-CTC、E-ACC/M-CTA、E-ACC/M-CTG、E-AGC/M-CTT、E-AGG/M-CTA6对标记了荧光的选择性引物进行PCR扩增,其产物在的CEQ8000遗传分析仪上检测。结果6对引物分别在罂粟、大麻和虞美人样本中检出27~46、5~20、4~31条扩增片段,种属间存在明显差异;同一罂粟根、茎、叶、花和果的DNA检测结果相同。结论罂粟、虞美人和大麻3种植物的AFLP分析结果显示出的差异性,同一植株不同部位DNA AFLP结果的同一性,有可能用于检测未知植物检材的种属来源。     

利用荧光AFLP技术检测罂粟DNA多态性

目的 利用荧光AFLP检测技术检测罂粟植物DNA多态性。方法 用Axygen公司的AxyPrep DNA试剂盒提取了12株产于缅甸和中国云南省昆明市宜良县罂粟植株的DNA，用Eco RI和Mse I对总DNA进行酶切。连接人工接头，预扩增和选择性扩增，其产物在CEQ8000遗传分析系统上检测。结果 64对选择性扩增引物中8对引物能得到20条以上的扩增片断，具有高度多态性。结论 荧光AFLP技术可用于罂粟DNA多态性的检测。     

AFLP分子标记技术的新进展及其在法医植物学中的应用

扩增片段长度多态性(amplified fragment length polymorphism,AFLP)是一种用来检测基因组多态性的新一代分子标记,具有分辨率高、稳定性好、重复性好等特点.近年来,研究人员对该技术进行了不断的优化和完善,并由之衍生出多种相关技术.AFLP技术在动物、植物及微生物等许多研究领域已有广泛应用,在法医植物学中得到初步发展并成为研究热点.本文主要介绍了AFLP技术的新进展以及在法医植物学中的应用情况.     

大麻的DNA分析检验技术

本文简要回顾了大麻的一些常规检验方法,并重点综述了大麻植物的DNA分析检验技术,包括随机扩增多态性DNA(RAPD)、测序扩增区段(SCAR)、短串联重复序列(STR)、扩增片段长度多态性(AFLP)、单核苷酸多态性(SNP)等DNA分子标记检测法。并且对这几种技术的应用范围及前景做了简要分析,以期为实践工作提供基础理论帮助。     

mtDNA—HVⅠ和细胞色素b片段的复合扩增及其法医学应用

目的探讨复合扩增mtDNA D环HV I和细胞色素b片段进行种属鉴定和个体识别的方法及mtDNA-HV I多态性。方法用两对引物同步扩增HV I片段与细胞色素b片段,银染显带检测扩增产物,ABI377测序仪及荧光测序技术分析扩增产物序列多态性。结果人类有279bp,358bp两条带,动物只有358bp一条带。通过对131例随机广东汉族人群个体进行mtDNA控制区(15997～16236))序列测定统计,得出此区域的序列多态性。共发现69个位点变异,平均每个个体存在2.679个碱基突变,检出67个单倍型,基因多样性为97.92％。结论mtDNA控制区(15997—16236)具有较高的序列多态性。为良好的个体识别标记。复合扩增mtDNA D环HV I与细胞色素b片段进行测序分析可以同步进行种属鉴定和个体识别。     

克隆牛的DNA鉴定

目的建立26个多态性STR标记,进行克隆牛的鉴定。方法采用荧光标记PCR引物,进行PCR扩增,应用GeneScan技术分析26个牛的微卫星基因座遗传多态性。结果26对荧光标记引物均能对上述样品很好地扩增出DNA产物,并得到清晰的条带。根据实验观察到的数据计算,体细胞克隆牛供体奶牛与克隆小奶牛的偶合率为3.31×10-15,认定它们为相同来源;在所检测的26个微卫星标记中,体细胞克隆牛供体奶牛、克隆小奶牛与克隆小奶牛代孕荷斯坦母亲的基因型在21个标记上不符合母子遗传关系,可以排除其亲子关系。结论有助于今后开展对珍稀动物或家畜被盗的案件鉴定工作。     

大麻遗传标记的法医学应用研究进展

大麻是大麻科大麻属一年生雌雄异株的草本植物,其内含有具有强烈成瘾性和麻醉性的四氢大麻酚(THC).大麻价格低廉、获取方便、且受到一些国家和地区合法化的影响,目前已成为滥用最广泛的毒品之一.因此,大麻植株的鉴定对于打击毒品犯罪、维护社会稳定具有重要意义.近年来,基于DNA遗传标记的大麻鉴定为案件侦破提供了新的技术手段,针...     

辽宁地区汉族群体15个STR基因座遗传多态性调查

本实验选用五色荧光标记引物复合扩增技术,对辽宁汉族群体15个STR基因座的遗传多态性进行调查,以期为法医学个人识别、亲权鉴定和群体遗传学研究提供基础数据。     

Genetic variation in hemp and marijuana (Cannabis sativa L.) according to amplified fragment length polymorphisms

Cannabis sativa L. (Cannabaceae) is one of the earliest known cultivated plants and is important in the global economy today as a licit and an illicit crop. Molecular markers distinguishing licit and illicit cultivars have forensic utility, but no direct comparison of hemp and marijuana amplified fragment length polymorphism (AFLP) has been made to date. Genetic variation was surveyed in three populations of fiber hemp and a potent cultivar of marijuana using AFLP markers. Ten primer pairs yielded 1206 bands, of which 88% were polymorphic. Eighteen bands represented fixed differences between all fiber populations and the drug cultivar. These markers have practical utility for (1) establishing conspiracy in the cultivation and distribution of marijuana, (2) identifying geographic sources of seized drugs, and (3) discriminating illegal, potent marijuana cultivars from hemp where the cultivation of industrial hemp is permitted.     

DNA polymorphism detection of Papaver somniferum L using fluorescent amplified fragment length polymorphism

OBJECTIVE: To detect DNA polymorphism of Papaver somniferum L using fluorescent Amplified Fragment Length Polymorphism. METHODS: Genomic DNA was isolated using the AxyPrep DNA Kit, double-digested by two restrictional endonucleases (Eco RI and Mse I) and ligated to oligonucleotide adapters. After Pre-amplification and selective amplification, the DNA fragments were separated by capillary electrophoresis using the CEQ8000 DNA Fragment Analyzer. RESULTS: More than 20 fragments of highly polymorphic products were obtained by 8 pairs of primer from 64 selective amplifying primer pairs. CONCLUSION: The fluorescent AFLP technique can be used to detect the DNA polymorphism of Papaver somniferum.     

Identification of Acer rubrum using amplified fragment length polymorphism

Amplified fragment length polymorphism (AFLP) analysis of botanical forensic evidence provides a means of obtaining a reproducible DNA profile in a relatively short period of time in species for which no sequence information is available. AFLP profiles were obtained for 40 Acer rubrum trees. Leaf material from five additional species was also typed. Genomic DNA was isolated using the DNeasy Plant Miniprep Kit (Qiagen, Valencia, CA), double-digested by two restriction endonucleases (EcoRI and MseI) and ligated to oligonucleotide adapters. Two consecutive PCR reactions (pre-amplification and selective amplification) were performed using a modification of the AFLP protocol described by Gibco (Invitrogen, Rockville, MD). The DNA fragments were separated by capillary electrophoresis using the CEQ 8000 DNA Fragment Analyzer. A number of Acer rubrum species-specific peaks were identified. In addition, within this closed set of samples, 15 of 16 (93.8%) blind samples were correctly identified. AFLP data can be used to determine the species of botanical evidence or to associate a sample to a source. This information can be used in forensic investigations to link a piece of evidence with a particular location or suspect.     

DNA profiling of disputed chilli samples (Capsicum annum) using ISSR-PCR and FISSR-PCR marker assays

A case of marketing of spurious seeds of chilli, Capsicum annum in the brand name of an elite variety referred to us from an Indian court of law, for identification is described here. The highly reproducible molecular marker assays, inter simple sequence repeat polymerase chain reaction [ISSR-PCR] and FISSR-PCR (for fluorescent ISSR-PCR) were used for differentiating the four disputed chilli samples. A total number of 17 ISSR anchored primers, which included nine di-, and eight tri-nucleotide primers were used for the analysis. The ISSR-PCR products were separated on a 2% agarose gel. A total of 212 and 288 bands were resolved by seven di- and eight tri-nucleotide primers, respectively, with an average of 30 bands per primer. Five out of nine dinucleotide primers and four out of eight trinucleotide primers could unambiguously differentiate all the four disputed chilli samples. The sensitivity and informativeness of the ISSR-PCR assay were further enhanced by the use of FISSR-PCR technique. The FISSR-PCR assay revealed a total number of 566 bands using three tri- and one di-nucleotide primers with an average of 141 bands per primer. These four primers could reliably distinguish all the four disputed samples unambiguously. In developing countries like India, violation of Plant Breeder's Rights is a major concern of law. The present report is, therefore, a step to protect the Plant Breeder's Rights by making use of reliable and modern DNA technologies.     

Analysis of human specificity in AFLP systems APOB,PAH, and D1S80

We have previously characterized and databased three human amplified fragment length polymorphism (AFLP) loci: the hypervariable regions 3′ to apolipoprotein B (APOB), phenylalanine hydroxylase (PAH) and at locus D1S80. The analysis utilized polymerase chain reaction (PCR) technology for human identification in forensic and paternity testing. This study extended that work by assessment of specificity of amplicons produced with non-human and human control DMAs for APOB, PAH and D1S80 under high and low stringency PCR conditions. It was seen that primate and other animal templates (with the exception of chimpanzee) yielded products below the human allele range under high stringency PCR parameters. Under reduced stringency PCR with animal and primate samples, reproducible genetic fingerprints were generated spanning the human allele range. The patterns were produced with defined human AFLP primer pairs under specifically relaxed PCR reaction and thermalcycling parameters. They showed genetic relationships between species at the DNA level. Amplicon patterns were compared for band size and intensity matches within the PCR synthesis range defined by the conditions used. This technique could become a useful tool in species identification and molecular evolutionary studies.     

Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay

Cannabis sativa is both an illegal drug and a legitimate crop. The differentiation of illegal drug Cannabis from non-drug forms of Cannabis is relevant in the context of the growth of fibre and seed oil varieties of Cannabis for commercial purposes. This differentiation is currently determined based on the levels of tetrahydrocannabinol (THC) in adult plants. DNA based methods have the potential to assay Cannabis material unsuitable for analysis using conventional means including seeds, pollen and severely degraded material. The purpose of this research was to develop a single nucleotide polymorphism (SNP) assay for the differentiation of "drug" and "non-drug"Cannabis plants. An assay was developed based on four polymorphisms within a 399 bp fragment of the tetrahydrocannabinolic acid (THCA) synthase gene, utilising the snapshot multiplex kit. This SNP assay was tested on 94 Cannabis plants, which included 10 blind samples, and was able to differentiate between "drug" and "non-drug"Cannabis in all cases, while also differentiating between Cannabis and other species. Non-drug plants were found to be homozygous at the four sites assayed while drug Cannabis plants were either homozygous or heterozygous.     

引物3'端第二个碱基SNP变异的长度多态性遗传标记检测

目的为了获得引物3'端存在SNP点突变的插入缺失遗传标记rs10644346所有等位基因片段。方法基于等位基因特异性PCR原理,设计一条共用上游引物,二条3’端倒数第二个位置特异性碱基的下游引物。运用该三条引物检测150个无关个体及10例亲子关系已确定的三联体,同时运用其中二条引物(共同上游引物和其中一条下游引物)扩增9例样本。结果三条引物扩增150个无关个体均有清晰扩增片段;10例三联体案例亲代与子代扩增片段均符合孟德尔遗传定律;二条引物扩增9例样本后发现特定片段丢失。结论本次研究设计的三条引物PCR,证明特异性碱基位置除了通常的3'末端,理想条件下3'端的其他位置(如倒数第二个位置)也可以成为有效选择,该三条引物设计方法为检测引物侧翼存在点突变的遗传标记提供了一种新的参考。     

Field testing of collection cards for Cannabis sativa samples with a single hexanucleotide DNA marker

The validity and feasibility of using DNA collection cards in the field for preservation and analysis of Cannabis sativa genotypes were investigated using a highly specific hexanucleotide marker. Collection cards were submitted to the National Marijuana Initiative, which selectively trained and managed the collection of specific types of samples from a variety of participating agencies. Samples collected at seizure sites included fresh marijuana leaf samples, dried "dispensary" samples, U.S. border seizures, and hashish. Using a standardized PCR kit with custom-labeled oligonucleotide primers specific to marijuana, collection cards produced eight genotypes and 13 different alleles, extremely low baselines, and no cross-reactivity with control plant species. Results were produced from all sample types with the exception of hashish. Plant DNA collection cards represent an easily implementable method for the genetic identification and relatedness of C. sativa street and grow site-seized samples with applications for databasing and market disruption.