Detection and identification of cannabis by DNA

The unambiguous identification of illicit substances, including Cannabis sativa, is a major concern of law enforcement agencies. Current methods of cannabis identification involve the use of techniques such as HPLC and GC to identify cannabinoids. A method for the identification of cannabis using DNA-specific primers has been developed and is described here. The nucleotide sequences between the trnL and trnF genes in the chloroplast of Cannabis sativa have been determined and Cannabis sativa-specific nucleotide sequences within the intergenic spacer between the trnL 3′ exon and trnF gene identified. Primers, made to these sequences, have been tested on a range of different plant extracts but only give a PCR product in the presence of Cannabis sativa. The successful production of a PCR product using these primers identifies the presence of cannabis.     

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。     

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.     

Organelle DNA haplotypes reflect crop-use characteristics and geographic origins of Cannabis sativa

Comparative sequencing of cannabis individuals across 12 chloroplast and mitochondrial DNA loci revealed 7 polymorphic sites, including 5 length variable regions and 2 single nucleotide polymorphisms. Simple PCR assays were developed to assay these polymorphisms, and organelle DNA haplotypes were obtained for 188 cannabis individuals from 76 separate populations, including drug-type, fibre-type and wild populations. The haplotype data were analysed using parsimony, UPGMA and neighbour joining methods. Three haplotype groups were recovered by each analysis method, and these groups are suggestive of the crop-use characteristics and geographical origin of the populations, although not strictly diagnostic. We discuss the relationship between our haplotype data and taxonomic opinions of cannabis, and the implications of organelle DNA haplotyping to forensic investigations of cannabis.     

纳米科技与法医DNA检验

综述了纳米科技进步将对法医DNA检验产生的深远影响,对从DNA提取到基因芯片研究等多个未来研究的新方向进行了探讨。     

Personal identification from human remains by mitochondrial DNA sequencing

The authors report four cases in which severely damaged human remains were identified by mitochondrial DNA (mtDNA) sequencing. Degraded DNA was extracted from highly adipoceratous tissues using the phenol-chloroform method and polymerase chain reaction amplified for sequencing of two hypervariable regions, hypervariable region 1 and hypervariable region 2, of mitochondrial DNA. They also sequenced these regions of blood samples that were obtained from the presumptive mother or sister of the human remains. The sequencing results were compared with each other and with the Anderson's sequence. It was concluded from the sequence data that a lower part of a body in case 1 and some organs in case 2 were from the same woman, and a human head in case 3 and a female body in case 4 were from the relative of a presumptive mother and a sister, respectively.     

DNA identification of sperm cells collected and sorted by flow cytometry

In cases of rape, obtaining enough biologic material for DNA identification of the attacker is often difficult because the methods for distinguishing and separating sperm cells from vaginal cells are not sufficiently efficacious. This article describes a new, innovative method for spermatic DNA extraction from the vaginal washing fluid by means of flow cytometry. The high specificity and sensitivity of the flow-cytometric sorting method provides enough sperm cells for DNA typing. The ease of execution of this method, involving vaginal washing with physiologic solution and flow-cytometric reading of the fresh sample, substantially increases its cost-benefit ratio.     

An unusual case of identification by DNA analysis of siblings

A badly decomposed body required identification by means of DNA analysis. A brother and sister of the deceased were available as reference subjects. Although investigation of Y-chromosomal markers established an exclusion condition, autosomal markers suggested a positive identification. In order to increase the reliability of the tests, X-chromosomal markers were also investigated. This analysis showed the body to have an XXY genotype (Klinefelter's syndrome). A number of hypotheses were assessed using biostatistical methods, ultimately resulting in a definite identification. The special aspect of Klinefelter's syndrome proved highly useful for biostatistical analysis.     

Sourcing Cannabis sativa L. by thermogravimetric analysis

Marijuana, dried and ground Cannabis, is the most consumed illicit drug in the world. Many undesirable and risky effects to human health are caused by its use. The medicinal use or legal recreational use of Cannabis has also been rising in many countries. These facts make traceability methodologies increasingly important whether for forensic use, such as drug trafficking eradication, or for quality control purposes of legal medicinal Cannabis. Consequently, the objective of this study was to analyze Cannabis by means of thermogravimetric analysis (TGA) in order to assess the capability of this technique to trace the geographical origin of Cannabis cultivated in Colorado, United States of America. TGA appears to be sensitive enough to detect the degradation/decarboxylation of cannabinoids and terpenes, at least to some extent; also, the degradation of cellulose, hemicellulose and lignin was indicated. Overall, the temperature ranges we analyzed using linear discriminant analysis showed high accuracies, with the 200 to 300 °C and 600 to 700 °C ranges achieving 100% accuracy.     

从蚊子血中检测人DNA

目的　从叮咬人后的蚊子体内血中得到被叮咬者的DNA分型。　方法　Chelex10 0法提取叮咬人后的蚊子血DNA ,PowerPlex 16试剂盒和Identifiler试剂盒扩增DNA。　结果蚊子血中可成功检测出被叮咬人的DNA基因型。　结论　可从蚊子血中检测到被叮咬人的DNA数据 ,结果稳定性好。两种试剂盒的联合应用 ,可大大提高检测的准确率和可靠性 ,对实际检案具有很大帮助。     

DNA identification in mass fatality incidents

DNA identification has become an important aspect of mass fatality management as well as in other instances of difficult identification of human remains. Most large mass fatality incidents will require DNA identification. Medical examiners should prepare for such potential eventuality. Whether DNA is tested, in mass fatality incidents, DNA specimens should be obtained from remains as well as from next-of-kin for potential testing. DNA identification is neither as slow nor as expensive relative to the overall fatality management as is commonly assumed. This article sought to provide medical examiners with a framework for DNA identification in mass fatality incidents.     

从蚊子血中检测人DNA

目的 从叮咬人后的蚊子体内血中得到被叮咬者的DNA分型。方法 Chelex100法提取叮咬人后的蚊子血DNA，PowerPlex16试剂盒和Identifiler试剂盒扩增DNA。结果 蚊子血中可成功检测出被叮咬人的DNA基因型。结论 可从蚊子血中检测到被叮咬人的DNA数据，结果稳定性好。两种试剂盒的联合应用，可大大提高检测的准确率和可靠性，对实际检案具有很大帮助。     

Big game species identification by deoxyribonucleic acid (DNA) probes.

Species identification is important in many big game forensic science cases but cannot always be accomplished because of the lack of adequate techniques. The authors have developed deoxyribonucleic acid (DNA) probes for elk, deer, and antelope by isolating highly repeated satellite sequences. These DNA probes distinguish among deer, elk, and antelope, although not between different species of deer. Because of the high number of sequence copies per genome, these probes are extremely sensitive, requiring less than 10 ng of total genomic DNA. The developmental protocol for these probes is relatively simple and is applicable to many other species.     

Variation in nuclear DNA concentrations during urination

This study examined the cellular origin and concentration of nuclear DNA in human urine. Ten subjects provided two entire, first-morning voids: one as a single specimen and one as a consecutive series of samples. The serial samples were centrifuged, organically extracted, and quantified by slot-blot analysis. Total DNA concentrations ranged from 0.02 to 21.3 ng/mL for the males and 25.0 to 96.9 ng/mL for the females. The female samples were found to contain numerous vaginal epithelial cells. DNA was detected in all of the serial samples of nine subjects; however, the DNA concentrations varied considerably. With six subjects, the DNA concentration of the first serial sample was at least three times greater than that of the entire void. DNA was only detected in the first 21% of the void from one male subject. The results of this study have implications for the collection of urine samples.     

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.     

Chiral identification and determination of ephedrine,pseudoephedrine, methamphetamine and metecathinone by gas chromatography and nuclear magnetic resonance

The enantiomers of the related substances methamphetamine, ephedrine, pseudoephedrine and methcathinone were determined by both gas chromatography after derivatization and by nuclear magnetic resonance using a chiral solvating agent. For GC the substances were derivatized with (R)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetic acid (MTPA) to give diasteromeric derivatives. Resolution (baseline) of at least 1.6 was obtained between all derivatives. NMR determination of the enantiomers was conducted in a chiral environment by the addition of the chiral solvating agent, (R)-(+)-1,1′-bi-2-naphthol, to NMR solutions of the substances. Racemization of methcathinone was demonstrated to be facile by exposure to alkaline solutions for varying periods of time. Enantiomeric ratios of some products derived from the oxidation of ephedrine were determined.     

Individual identification from semen by the deoxyribonucleic acid (DNA) fingerprint technique

For individual identification from semen, the deoxyribonucleic acid (DNA) fingerprint technique was used. In a blind trial, we succeeded in determining the semen donors among several volunteers comparing the DNA fingerprints of the blood and semen samples, respectively. Thereafter, we examined semen in a condom left beside a naked female dead body. The DNA fingerprint of the semen was recognized to be identical to that of the blood from a suspected man arrested later. This is the first report that the DNA fingerprint technique was practically used in a criminal investigation in Japan.     

TUNEL法检测大鼠死后心肌细胞核DNA断裂与死亡时间关系研究

目的用原位脱氧糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)观察大鼠死后心肌细胞DNA断裂与死亡时间(postmortem interval,PMI)的关系。方法建立SD大鼠死亡模型,在25.1℃,分别于死后6、12、24、36、48、60h提取大鼠心肌组织进行TUNEL检测,显微成像系统采集图像,经计算机图像系统分析及统计学处理。结果死后6~60h,心肌细胞核荧光染色依次由绿色、黄绿色亮度增强、亮黄色到荧光亮度减弱;形态由扁圆形或柱状,大小较均匀,到体积增大、呈毛虫状、多数核碎裂到形态不规整,大部分核形成碎片;心肌组织细胞核的IG,死后6h至36h依次增高,36h达到高峰,随后下降;心肌细胞核DNA的AG死后24h最高。结论死后各时间点,心肌细胞核的荧光颜色和亮度以及细胞核的大小和形态有一定的变化规律。     

Evaluation and quantification of nuclear DNA from human telogen hairs

Nuclear DNA was extracted from human telogen hairs from 60 individuals. Six to nine hairs from each individual were individually extracted. The amount of DNA recovered from each individual varied greatly, and most samples yielded a quantity of 550 pg or less per hair. A selective extraction buffer was used to remove epithelial cell DNA and the amount of exogenous DNA was determined. DNA was also quantified by real time PCR using three different sized amplicons targeting an Alu sequence. The results were used to determine the state of degradation of the extracted DNA. Different quantities of sample (<100 pg, 100-500 pg, >500 pg) were amplified with the Miniplex kits to determine the minimum DNA template required for successful amplification. DNA recovered from hair showed degradation; however, partial profiles were obtained for those samples containing at least 60 pg using MiniSTRs.