应用AFLP检测大麻遗传多样性

目的 筛选对于大麻多态性好的AFLP引物标记来区分大麻品种.方法 利用AFLP技术用55对引物组合对12个大麻地域品种进行初筛.结果 选出5对多态性好的引物组合进行了遗传多样性研究.每对AFLP引物组合扩增出47～76务带.共获得285条带,其中多态性条带为99条以及10条品种特异带.结论 AFLP对于大麻具有很高的分辨率,为今后深入地研究大麻植物遗传多样性奠定了很好的基础.     

RAPD analysis distinguishes Cannabis sativa samples from different sources

Samples of the seeds and seedlings of Cannabis sativa, and its dried leaves and flowerheads (marijuana), could be reliably distinguished by RAPD-PCR (Random Amplified Polymorphic DNA using the Polymerase Chain Reaction). DNA was best extracted from fresh tissues using buffers and the detergent cetyltrimethylammonium bromide; poorly dried tissue or inviable seed yielded coloured samples of degraded DNA. DNA was isolated from 51 C. sativa and two Humulus lupulus (hops) samples. Of the C. sativa samples 43 were from Australia (ten from Canberra gardens, eight from a New South Wales crop and 25 from two Queensland crops) and eight were from Papua-New Guinea (P-NG). A total of 102 different bands were obtained using four 10-nucleotide primers with arbitrarily chosen sequences. Banding patterns were compared by calculating pairwise distances using various algorithms, and presented using the neighbour-joining tree and multidimensional scaling methods. These showed a clear difference between C. sativa and H. lupulus, and separated the samples of the latter into three distinct groups; one group comprised all the P-NG samples, another the Canberra samples, and the third, the three crop samples.     

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。     

Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region

Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog, goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic specimens.     

New alleles and mutational events at 14 STR loci from different German populations

The molecular origin of DNA mutations and the mutation rates were analyzed at 14 short tandem repeat (STR) loci with samples from trio cases derived from 10 different German population samples. STR loci comprised of D2S1360, D3S1744, D4S2366, D5S2500, D6S474, D7S1517, D8S1132, D10S2325, D12S391, D18S51, D19S246, D20S480, D21S226, and D22S689. In a total of 488 meioses, 16 isolated genetic inconsistencies in 8 different STRs were observed, whereas no mutations were found at the other loci. The data of five mutations suggested the presence of silent or null alleles due to sequence variation in primer binding site. This could be confirmed for four suspected cases by the use of alternative primer sets and by DNA sequence analyses. Furthermore, this study revealed nine new allelic variants at five different loci.     

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.     

Industry-sponsored medical education--in the quest for professional integrity and legal certainty

Industry-sponsored medical education is a much disputed issue. So far, there has been no regulatory framework which provides clear and definite rules as to whether and under what circumstances the sponsorship of medical education is acceptable. State regulation does not exist, or confines itself to a very general principle. Professional regulation, even though applied frequently, is rather vague and indefinite, raising the general question as to whether self-regulation is the right approach at all. Certainly, self-regulation by industry cannot and should not replace other regulatory approaches. Ultimately, advertising law in general and the European Directive 2001/83/EC specifically, might be a good starting point in providing legal certainty and ensuring the independence of medical education. Swiss advertising law illustrates how the principles of the European Directive could be implemented clearly and unambiguously.     

Locked nucleic acids: Increased trace DNA amplification success with improved primers

Locked nucleic acids (LNAs) are a conformationally restricted DNA analog, which can be incorporated into oligonucleotides to increase binding strength. To investigate if LNAs increase amplification success for trace DNA samples in a forensic context, primer sequences for four routinely used STR loci (FGA, D7S820, D13S317 and D18S51) have been altered to include LNA bases. The LNA modified primers display a broader tolerance to a range of reaction conditions compared to unmodified DNA primers, with higher T_ms giving increased specificity. Increased peak heights, improved peak height ratios and decreased template requirements were seen with LNA primers. The increased amplification success of LNA primers, and broader range of optimal reaction conditions, suggest that using LNA primers for multiplex STR genotyping assays could be highly beneficial for trace DNA genotyping.     

Quantification of trace amounts of human and non-human mitochondrial DNA (mtDNA) using SYBR Green and real time PCR

There are currently no tests available to quantify total non-human mammalian mtDNA. Standard universal DNA quantification tests are unsuitable due to the large size difference between nuclear and mitochondrial genomes and the ubiquity of human mtDNA. A method has therefore been developed to quantify total mammalian mtDNA and total human mtDNA present in a sample using SYBR Green.Mammalian primers designed to react with all mammals were designed on the 12S and human specific primers were designed on the cytochrome b gene. Each primer set was reacted separately with sample and SYBR Green and detected using RT-PCR. A standard curve was developed using dilutions ranging from 1 billion copies to 100 copies of mtDNA.Twenty-four human samples were analysed and an average log (copy number) human/universal ratio of 1.00 was obtained. Samples falling below this ratio will contain some non-human mtDNA while samples falling above this ratio contain human mtDNA only.Twenty-nine mammal samples were also tested. 96.6% of these showed human contamination to some extent. This test is able to quantify mtDNA down to the femtogramme (10E−15g) level.     

Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

Psilocybe cubensis, or “magic mushroom,” is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web‐based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy^® Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR^® Green I, Radiant? Green, or LCGreen Plus^® intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR^® Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant? Green dye.     

Detection of species of graft in xenotransplants using arbitrary primed polymerase chain reaction.

Because of a shortage in the availability of human organs, xenografts have been attempted in humans with cardiac, renal, and hepatic failure, despite limited success. Use of xenografts, however, is regulated under law in various countries. In xenotransplant cases related to violation of transplantation law, determination of species of the source of tissue and organ(s) becomes highly essential. Random amplified polymorphic DNA (RAPD) protocols using six sets of arbitrary short-sequenced primers have been standardized for verifying claims of porcine cardiac and renal grafts in human transplantation cases. Six arbitrary primers used were found to generate unique amplicon patterns at 36 degrees C annealing temperature. Among the selected primers, a single primer set having the sequence 5'- GGTGCGGGAA -3' is found to be the most informative in discerning porcine tissue contamination in humans. The patterns obtained were consistent for a particular genome. The grafted organs in the studied case were analyzed to be of porcine origin.     

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。     

Identification of a D8S1179 primer binding site mutation and the validation of a primer designed to recover null alleles

A population study of Chamorros and Filipinos using short tandem repeat (STR) loci amplified with the AmpFlSTR Profiler Plus PCR amplification kit demonstrated an excess of observed homozygosity at the D8S1179 locus. Use of a different set of D8S1179 primers to type the same samples did not demonstrate an excess of homozygosity and showed discordant genotypes at the D8S1179 locus. A single point mutation, G-to-A transition, 16 nucleotides from the 3' end of the reverse primer, was identified to cause allele dropout when using the AmpFlSTR Profiler Plus primer set. An additional D8S1179 reverse primer specific for the variant was constructed resulting in the recovery of the null allele. The primer was included in the newly developed AmpFlSTR Identifiler PCR amplification kit. No deleterious effects or non-specific peaks were observed in validation experiments evaluating primer concentration, Mg2+ concentration, annealing temperature and population samples.     

Assessment of disputed identity of blood alcohol samples using DNA fingerprinting

DNA fingerprinting is a perfect tool for investigating the identity of disputed blood by alcohol samples extracted. However, blood samples stored at an ambient temperature for longer periods can show considerable degradation of high-molecular DNA, diminishing the value of fingerprint investigation because of loss of the less frequent bands formed by the longer DNA fragments. Addition of the complexing agent EDTA can retard this degradation. Determination of the sex with DNA probes in the blood alcohol sample increases confidence in the investigation.     

纽伦堡审判在当代国际人权保护中的作用

纽伦堡审判犹如当代国际人权法发展历程上的里程碑，它将自然法思想引入到以法律实证主义为基础的实在法中，突破了国家主权在国际人权保护中形成的障碍，明确了个人应承担的国际法责任与义务，使个人成为国际法的不法主体，进而使法律的“应然”与“实然”结合在一起。联合国发扬光大了纽伦堡审判的精神和实质，使国际人权法成为国际法中的一个重要分支，从纽伦堡审判到《国际刑事法院规约》的生效，后者目前所遇到的问题与60年前的情况相似，它是否能如设计者所希望的那样还需大国在其中发挥更大的作用。     

邓恩伯格论私法体系及债权物权的区分

邓恩伯格将私法权利主要分为财产权与关于人的权利;其中财产权包括了债权与物权,而继承权虽然并非财产权,但也是财产移转的方式,因此也在此论述;关于人的权利则包括人格权与亲属权。与此相应,其私法体系由总则、物权法、债法、人法———亲属法、继承法五部分组成。他还将债权与物权的区分主要建立在客体的不同之上。可以看出,邓恩伯格比较忠实地继承了萨维尼所构造的体系。     

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。     

A study on the effects of degradation and template concentration on the amplification efficiency of the STR Miniplex primer sets

In forensic DNA analysis, the samples recovered from the crime scene are often highly degraded leading to poor PCR amplification of the larger sized STR loci. To avoid this problem, we have developed STR markers with redesigned primer sequences called "Miniplexes" to produce smaller amplicons. To assess the effectiveness of these kits, we have tested these primer sets with enzymatically degraded DNA and compared the amplifications to a commercial kit. We also conducted sensitivity and peak balance studies of three Miniplex sets. Lastly, we report a case study on two human skeletal remain samples collected from different environmental conditions. In both types of degraded DNA, the Miniplex primer sets were capable of producing more complete profiles when compared to the larger sized amplicons from the commercial kit. Correct genotypes were obtained at template concentrations as low as 31 pg/25 microL. Overall, our data confirm that our redesigned primers can increase the probability of obtaining a usable profile in situations where standard kits fail.     

片段长度差异等位基因特异性PCR——一种改良的SNP分型新方法

目的建立一种基于等位基因特异性PCR原理的改良SNP分型新方法:片段长度差异等位基因特异性PCR,并考察特异性引物的3'端第3位、第4位碱基错配对特异性延伸的影响。方法以SNP位点rs759117和rs760887为例,设计两条长度不同、3'末端分别与SNP两个等位基因碱基配对的上游引物,同时在两个等位基因特异性引物3'端第3或第4位碱基引入错配以增加特异性,下游为公用引物。PCR产物经聚丙烯酰胺凝胶电泳、银染显带后确定样本的基因型。结果不同SNP纯合子为长度不同的单一谱带,杂合子则为两条带,其结果与直接测序完全一致。两条特异性上游引物3'端第3或第4位碱基引入错配后非特异性延伸显著减少,且对PCR反应条件的严格性要求明显降低。结论片段长度差异等位基因特异性PCR是一种简单快速而有效的SNP分型新方法;两条特异性引物3'端第3、第4位碱基引入错配可使特异性显著增加     

论《旅游权利法案》的国际法性质及在我国的适用

《旅游权利法案》是世界旅游组织制定的规范性文件，属习惯国际法，可通过国家实践和法律确信在内国得到适用。我国在制定旅游基本法时应转化和吸纳其相关原则、制度与规范，以国家立法形式使《旅游权利法案》在我国得到适用。