S. Rajaei, Rana Sabagh Farshi, Maryam Moazzam Jazi, S. Seyedi
{"title":"黄连木根DNA提取过程中酚类化合物的有效去除策略。","authors":"S. Rajaei, Rana Sabagh Farshi, Maryam Moazzam Jazi, S. Seyedi","doi":"10.17503/agrivita.v39i3.734","DOIUrl":null,"url":null,"abstract":"Optimization of DNA extraction protocols for plant tissues and including endophytic microorganisms is a critical step of advanced plant-microbe interaction in agricultural studies. Pistachio ( Pistacia vera L.) root tissue contains high levels of polyphenols have been known as major extract contaminants and inhibitors of enzymatic activities during amplification. The present study aimed to develop reliable strategies to purify DNA from Pistachio root samples. Inhibiting substances were removed from DNA through a process including extraction with hot detergent contains SDS-Tris- EDTA, AlNH4(SO4)2.12H2O as chemical coagulating factor and CTAB-NaCl. Following typically organic extraction/alcohol precipitation, denaturing agarose electrophoresis performed to purify probable remain contaminants. The purified DNA was enough free of polyphenols based upon loss of color and spectral quality (260/230>1.6) and efficiently amplified during polymerase chain reaction particularly in the present of GC-clamp primers. This method proved well with detection of Glomus sp. (arbuscular mycorrhiza fungi) associated with Pistacia vera L. using denaturing gradient gel electrophoresis (DGGE).","PeriodicalId":402006,"journal":{"name":"Agrivita : Journal of Agricultural Science","volume":"1 1","pages":"0"},"PeriodicalIF":0.0000,"publicationDate":"2017-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":"{\"title\":\"Efficient Strategies for Elimination of Phenolic Compounds During DNA Extraction from Roots of Pistacia vera L.\",\"authors\":\"S. Rajaei, Rana Sabagh Farshi, Maryam Moazzam Jazi, S. Seyedi\",\"doi\":\"10.17503/agrivita.v39i3.734\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Optimization of DNA extraction protocols for plant tissues and including endophytic microorganisms is a critical step of advanced plant-microbe interaction in agricultural studies. Pistachio ( Pistacia vera L.) root tissue contains high levels of polyphenols have been known as major extract contaminants and inhibitors of enzymatic activities during amplification. The present study aimed to develop reliable strategies to purify DNA from Pistachio root samples. Inhibiting substances were removed from DNA through a process including extraction with hot detergent contains SDS-Tris- EDTA, AlNH4(SO4)2.12H2O as chemical coagulating factor and CTAB-NaCl. Following typically organic extraction/alcohol precipitation, denaturing agarose electrophoresis performed to purify probable remain contaminants. The purified DNA was enough free of polyphenols based upon loss of color and spectral quality (260/230>1.6) and efficiently amplified during polymerase chain reaction particularly in the present of GC-clamp primers. This method proved well with detection of Glomus sp. (arbuscular mycorrhiza fungi) associated with Pistacia vera L. using denaturing gradient gel electrophoresis (DGGE).\",\"PeriodicalId\":402006,\"journal\":{\"name\":\"Agrivita : Journal of Agricultural Science\",\"volume\":\"1 1\",\"pages\":\"0\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2017-08-07\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Agrivita : Journal of Agricultural Science\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.17503/agrivita.v39i3.734\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Agrivita : Journal of Agricultural Science","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.17503/agrivita.v39i3.734","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 1
摘要
优化植物组织和内生微生物的DNA提取方案是农业研究中植物-微生物高级相互作用的关键步骤。开心果(Pistacia vera L.)根组织中含有高水平的多酚,已被认为是主要的提取物污染物和酶活性的抑制剂。本研究旨在建立可靠的策略,从开心果根样品中纯化DNA。采用以SDS-Tris- EDTA、AlNH4(SO4)2.12H2O为化学凝固因子、CTAB-NaCl为溶剂的热洗涤剂进行萃取,去除DNA中的抑制物质。在典型的有机萃取/酒精沉淀后,进行变性琼脂糖电泳以纯化可能残留的污染物。根据颜色和光谱质量的损失(260/230>1.6),纯化的DNA足够不含多酚,并且在聚合酶链反应中有效扩增,特别是在目前的GC-clamp引物中。用变性梯度凝胶电泳(DGGE)检测与黄连木相关的丛枝菌根真菌Glomus sp.效果良好。
Efficient Strategies for Elimination of Phenolic Compounds During DNA Extraction from Roots of Pistacia vera L.
Optimization of DNA extraction protocols for plant tissues and including endophytic microorganisms is a critical step of advanced plant-microbe interaction in agricultural studies. Pistachio ( Pistacia vera L.) root tissue contains high levels of polyphenols have been known as major extract contaminants and inhibitors of enzymatic activities during amplification. The present study aimed to develop reliable strategies to purify DNA from Pistachio root samples. Inhibiting substances were removed from DNA through a process including extraction with hot detergent contains SDS-Tris- EDTA, AlNH4(SO4)2.12H2O as chemical coagulating factor and CTAB-NaCl. Following typically organic extraction/alcohol precipitation, denaturing agarose electrophoresis performed to purify probable remain contaminants. The purified DNA was enough free of polyphenols based upon loss of color and spectral quality (260/230>1.6) and efficiently amplified during polymerase chain reaction particularly in the present of GC-clamp primers. This method proved well with detection of Glomus sp. (arbuscular mycorrhiza fungi) associated with Pistacia vera L. using denaturing gradient gel electrophoresis (DGGE).