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标题：Testing of DNA isolation for the identification of hemp
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作者：Tomáš Vyhnánek ; Václav Trojan ; Klára Štiasna 等
期刊名称：Potravinarstvo : Scientific Journal for Food Industry
印刷版ISSN：1338-0230
电子版ISSN：1337-0960
出版年度：2015
卷号：9
期号：1
页码：393-397
DOI：10.5219/509
语种：English
出版社：Association HACCP Consulting
摘要：Hemp is diploid organism (2n = 2x = 20, genome size 534 Mb) with nine pairs of autosomes plus XX (♀) or XY (♂) chromosomes. Cannabis sativa L. is an important economic plant for the production of food, fibre, oils, and intoxicants. Genotypes (varieties or chemovar) of hemp with low Δ⁹-tetrahydrocannabinol content are used for industrial applications. Varieties with high Δ⁹-tetrahydrocannabinol or high cannabidiol content are used for medicinal applications. Biochemical and molecular methods can be used for identification and classification. An important step for molecular biology methods is to obtain the matrix of the native and sufficiently pure DNA. We tested two different experimental variant of samples (20 mg and 100 mg) of seeds, oilcake and dried flowers for analysis of the Italian variety Carmagnola for analysis (harvested in 2014, Hempoint Ltd., Czech Republic). The DNeasy® Plant Mini Kit (Qiagen, GE) was used to isolate the DNA. The DNA concentration and purity was assessed by agarose electrophoresis and via a spectrophotometer. Samples of lower weight yielded lower values of DNA concentration (average 16.30 - 38.90 ng.µL^-1), but with better purity than samples of higher weight (ratio A_260nm/A_280nm for low-weight samples was near 1.80). To test the applicability of DNA analysis, we used two SSR markers ( CAN1347 and CAN2913 ). PCR products were separated on 1% agarose and on 8% polyacrylamide electrophoresis. DNA samples obtained from samples of higher weight exhibited less PCR amplification than samples of lower weight. We found no effect of sample weight on the formation of non-specific amplification products during the PCR reaction. Based on our results we can be recommended for practical isolation procedure using DNeasy® Plant Mini Kit with lower of sample weight (20 mg). In future work the procedure for DNA isolating from wheat-cannabis products, e. g. breads, rolls or pasta, will be optimized.
其他摘要：Hemp is diploid organism (2n = 2x = 20, genome size 534 Mb) with nine pairs of autosomes plus XX (♀) or XY (♂) chromosomes. Cannabis sativa L. is an important economic plant for the production of food, fibre, oils, and intoxicants. Genotypes (varieties or chemovar) of hemp with low Δ9-tetrahydrocannabinol content are used for industrial applications. Varieties with high Δ9-tetrahydrocannabinol or high cannabidiol content are used for medicinal applications. Biochemical and molecular methods can be used for identification and classification. An important step for molecular biology methods is to obtain the matrix of the native and sufficiently pure DNA. We tested two different experimental variant of samples (20 mg and 100 mg) of seeds, oilcake and dried flowers for analysis of the Italian variety Carmagnola for analysis (harvested in 2014, Hempoint Ltd., Czech Republic). The DNeasy® Plant Mini Kit (Qiagen, GE) was used to isolate the DNA. The DNA concentration and purity was assessed by agarose electrophoresis and via a spectrophotometer. Samples of lower weight yielded lower values of DNA concentration (average 16.30 - 38.90 ng.µL-1), but with better purity than samples of higher weight (ratio A260nm/A280nm for low-weight samples was near 1.80). To test the applicability of DNA analysis, we used two SSR markers (CAN1347 and CAN2913). PCR products were separated on 1% agarose and on 8% polyacrylamide electrophoresis. DNA samples obtained from samples of higher weight exhibited less PCR amplification than samples of lower weight. We found no effect of sample weight on the formation of non-specific amplification products during the PCR reaction. Based on our results we can be recommended for practical isolation procedure using DNeasy® Plant Mini Kit with lower of sample weight (20 mg). In future work the procedure for DNA isolating from wheat-cannabis products, e. g. breads, rolls or pasta, will be optimized.
关键词：Cannabis; seed; oilcake; dry flower; DNA isolation
其他关键词：Cannabis;seed;oilcake;dry flower;DNA isolation