川芎DNA提取方法的研究

目的 以伞形科蒿本属植物川芎Ligusticum chuanxiong Hort.叶为材料,研究川芎DNA的提取方法 .方法 分别采取CTAB法、高盐低pH法、木本植物DNA提取法、改良高盐低pH法提取基因组DNA,并对4种方法进行了改进.通过0.9%琼脂糖凝胶电泳和RAPD两种方法检测所提取的DNA样品.结果 比较DNA产量、质量等,确定了木本植物DNA提取法最佳.结论 木本植物DNA提取法为最佳方法.     

贝母的分子生物学鉴定方法的研究

用ＤＮＡ指纹图谱、ＲＡＰＤ和ＤＮＡ测序等技术鉴定中药材已有不少报道[1～ 5] 。ＤＮＡ的提取和ＰＣＲ扩增是分子生物学最基本的操作步骤之一。但是由于商品药材多数经过加工处理 ,ＤＮＡ可能会受到不同程度的降解 ,再者植物药材中含有的次生代谢产物如多糖很难与ＤＮＡ和ＲＮＡ分开 ,而多糖对酶有抑制作用 ,因此将会影响ＤＮＡ的提取和ＰＣＲ扩增。本文以贝母为研究对象 ,探讨不同ＤＮＡ提取方法、不同扩增条件对ＰＣＲ产物的影响 ,为生药的分子生物学鉴定提供参考。材料与方法1　材料　托里贝母Ｆｒｉｔｉｌｌａｒｉａｔｏｒｔｉｆｏｌ…     

贝母属药用贝母总DNA提取方法的比较研究

目的 比较筛选最适于贝母属药用贝母的总DNA提取方法。方法 利用试剂盒法、改良的CTAB法、改良的SDS法提取贝母属药用贝母的总DNA;通过核酸蛋白检测仪及琼脂糖凝胶电泳方法检测其浓度和纯度;进行ISSR-PCR扩增检测所得总DNA质量。结果 3种方法均可提取出产量较高的基因组DNA,但试剂盒法相较提取的总DNA纯度最高,质量最好,并且均能扩增出丰富清晰的条带,稳定性高,操作简单耗时短。改良的CTAB法、SDS法提取的总DNA纯度、质量均次于试剂盒法,操作复杂耗时长,不适用于下游的分子生物学实验。结论 试剂盒法为贝母属药用贝母总DNA提取的最佳方法,其所得样品适用于PCR及其他分子生物学研究。     

牡丹皮基因组DNA提取的影响因素研究

目的:研究牡丹皮基因组DNA提取的影响因素。方法:以根皮类中药材牡丹皮为材料,在快速少量抽提(CTAB)法的基础上对提取缓冲液中氯化钠、β-巯基乙醇浓度,水浴温度、时间,RNA酶(RNaseA)、聚合酶链(PCR)反应体系等条件进行考察。结果:使用经过改进后的CTAB法获得的DNA纯度和完整性较好,A260/A280值在1.8～2.0之间,PCR扩增条带清晰且亮,从而为接下来的分子生物学实验打下了良好的基础。结论:本方法经济、快速、高效,可作为根皮类中药材基因组DNA的提取方法,可为大规模生产提供理论依据。     

一种适用于聚合酶链反应的常见念珠菌基因组DNA提取方法的研究

目的介绍一种简单、快速的真菌DNA提取方法,提高真菌DNA提取效率,减少毒性和污染性,以适应临床研究需要。方法同时用溶细胞酶结合Biospin真菌基因组DNA提取试剂盒和十六烷基三甲基溴化铵(CTAB)法,提取白色念珠菌、热带念珠菌、克柔念珠菌和净平滑念珠菌基因组DNA,用A260/A280的比值检测DNA的纯度并计算质量浓度,同时行聚合酶链反应(PCR)以评价其可靠性。结果溶细胞酶结合Biospin真菌基因组DNA提取试剂成功提取所用真菌基因组DNA,其纯度及质量浓度能满足PCR反应的要求。结论用溶细胞酶结合Biospin真菌基因组DNA提取试剂提取DNA,简单、快速、高效,可用于PCR反应。     

玉竹基因组DNA的提取与分析

目的以玉竹根茎为材料,寻找一种适合于玉竹总DNA提取的方法,便于下游分子生物学的操作。方法采用经典CTAB法与改良CTAB法。结果改良CTAB法提取得到的DNA可以很好地用于PCR扩增。结论改良CTAB法适用于类似玉竹这类含多糖较多的根及根茎类中药材DNA的提取。     

药用百合鳞叶中总DNA提取方法的研究

目的:对药用百合DNA的提取过程进行改良和优化,筛选出较理想的DNA提取方法。方法:在传统十六烷基三甲基氯化铵(CTAB)提取方法基础上,进行改良和优化,寻找出一种实用的DNA提取方法。结果:用改良CTAB法提取的总DNA的OD260/OD280在1.6~2.0之间,电泳条带清晰,其含量和纯度完全满足引物扩增分析的要求。结论:本试验中的百合鳞叶总DNA的提取方法简便实用,所得DNA的产量和纯度均能满足基因工程操作的要求。     

白木香基因组DNA的提取及AFLP银染体系的建立

用改良的CTAB法提取不同居群的白木香基因组DNA，通过优化AFLP技术中关键步骤的参数，建立适合白木香的AFLP银染反应体系。结果表明，改良的CTAB法提取的白木香基因组DNA纯度高，完整性好，满足AFLP分析的要求；250ng的基因组DNA用EcoRI和MseI37℃消化3h后可以被完全酶切，酶切产物和接头经16℃连接过夜后，用带有一个选择性碱基的引物和带有3个选择性碱基的引物分别进行预扩增和选择性扩增，扩增产物经变性聚丙烯酰胺凝胶电泳分离，用AgNO3染色得到了清晰的指纹式样，为下一步的遗传分析奠定了基础。     

Comparason of extraction methods for PCR detection of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> complex (BCC) from cystic fibrosis patients

Direct detection of Burkholderia cepacia complex (BCC) and its genomovars from sputum by molecular tests emerges as a method for rapid identification. In this study, four DNA extraction methods were evaluated for the identification for BCC from sputum of CF patients. Sputa from 28 CF patients were aliquoted and spiked with BCC reference strain. Boiling, phenol-chloroform, CTAB methods and a commercial spin column kit was used for DNA extraction. Total DNA yields were determined by spectrophotometry and single-round recA PCR was used for detection of BCC. No significant difference was observed in DNA yields from different extraction methods. Lower limit of detection for recA PCR was determined as 10⁶ cfu/ml. Amplification was observed in 7/16 (43.7%) of sputa for boiling, 8/16 (50%) of sputa for CTAB and 13/16 (81.2%) of sputa for phenol-chloroform method and spin column kit in the assay sensitivity range determined in the study. Phenol-chloroform and commercial spin column kit were found to be better suited for DNA purification from sputum of CF patients for BCC identification. Diagnostic impact of single-round recA PCR directly from sputum was limited to chronically-infected patients.     

在干细胞药物临床前研究中动物样品基因组DNA高通量提取的固相萃取法和磁珠筛选法初探

干细胞药物临床前研究中,常需要以干细胞特定基因片段为靶点追踪其体内的分布过程,需要提取动物样品基因组DNA,涉及到心脏、肝脏、肺脏、脾脏、肾脏、脑、骨髓、血、脂肪、肌肉、性腺、胃和肠等多种组织类型。为实现临床前样品的高通量检测,总结动物组织基因组DNA提取方法,并就如何高效高质提取样品基因组DNA进行讨论,提出间充质干细胞临床前研究中样品处理的一种可行的方案：通过生物样品制备仪获取稳定均一的组织裂解液,利用固相萃取法和磁珠筛选法配合使用获得高质量高浓度的动物样品DNA。     

丝状真菌顶头孢霉染色体DNA的提取

目的探索提取头孢菌素C产生菌顶头孢霉基因组DNA的方法。方法采用氯化苄法、液氮研磨法和酶裂解法。结果较其他两种方法比 ,氯化苄法提取效果好。当提取液pH值为 9 0～10 0、EDTA浓度为 12 0mmol·L-1时 ,所得的顶头孢霉染色体DNA的质量和数量均较理想 ,每克(湿重 )顶头孢霉菌丝体能得到 96 μg的染色体DNA。常规琼脂糖凝胶电泳分析 ,其DNA片断大于2 3kb。结论该分离方法获得的染色体DNA质量较高 ,可进行限制性内切酶酶解并适合后续特定基因的PCR扩增反应     

芋螺基因组DNA提取方法的优化

热带药用海洋生物芋螺产生的芋螺毒素(Conotoxin),已成为神经科学研究的有力工具药和新药开发的新来源。近来出现了从芋螺基因组DNA中克隆新型芋螺毒素基因的新方法．因而快速获得芋螺基因组DNA是分离多样性毒素基因、建立基因库及药用芋螺基因资源开发的前提。本研究采用不同的DNA提取方法．从6种芋螺的不同组织和器官中分离总DNA，经综合比较，建立了简便快速的提取芋螺总DNA的改良苯酚-SDS优化方法。     

Development of a quantitative PCR assay for residual mouse DNA and comparison of four sample purification methods for DNA isolation

Reliable and sensitive assays are required to determine whether a pharmaceutical product meets current regulatory guidelines for residual host cell DNA. In this study, the sensitivity of the qPCR assay was significantly improved by targeting the repetitive elements of mouse genome. This improved method allowed for sensitive and accurate quantitation of mouse genomic DNA in the range of 1 to 10⁶ pg/mL. In addition, four sample purification methods for DNA isolation (Wako DNA extractor kit, MasterPure™ DNA purification kit, PrepSEQ™ residual DNA sample preparation kit, and phenol–chloroform extraction method with addition of glycogen), each representing a different strategy for DNA isolation from proteinaceous solutions, were evaluated by isolating DNA from a mouse monoclonal IgG antibody. Among these methods, Wako DNA extractor kit and MasterPure™ DNA purification kit demonstrated superior DNA recovery, repeatability, and sensitivity, with quantitation limits of 1 pg/mL. To further evaluate these two DNA isolation methods, six replicates of an unspiked mouse polyclonal IgG antibody sample were tested by both methods, and both methods demonstrated a good degree of precision. Therefore, the residual mouse DNA quantitation methods described here represented rapid, accurate, precise, and sensitive procedures that can be used in quality control testing for regulatory compliance in the pharmaceutical industry.     

三种全血基因组DNA提取方法的比较

目的比较高盐法、改良高盐法和超声波处理法三种方法对全血基因组DNA的提取效果。方法取200ul肝素抗凝血，分别采用上述三种方法提取基因组DNA，用紫外分光光度计和琼脂糖凝胶电泳检测其纯度和含量。结果三种方法提取的基因组DNA含量分别为：高盐法38ug／ml全血；改良高盐法76．5ug／ml全血；超声波处理法20ug/ml全血；三种方法提取的基因组DNA纯度用A260／A280表示分别为：高盐法1．62；改良高盐法1．46；超声波处理法4．10。结论三种全血基因DNA提取方法操作简便、安全、快速，以改良高盐法提取效率最高。     

结直肠癌患者粪便细菌DNA提取方法的比较研究

摘要：目的比较结直肠癌患者粪便细菌DNA提取方法的优缺点及获得DNA的质与量,有利于结直肠癌患者肠道相关分子微生态的研究。方法采用传统法,化学裂解法和TIANamp bacteria DNA Kit试剂盒法,提取粪便中细菌DNA,比较其作为模板,用于细菌16SrRNA基因PCR扩增后凝胶电泳的优缺点及用核酸蛋白检测仪Biophotmeter测定提取DNA的质与量。结果传统法提取DNA的量及纯度均较低（22．37±2．45,3．24±0．22）,化学裂解法和试剂盒方法提取的量及纯度均较高,（分别为33．87±2．06,2．18±0．44;38．60±9．00,1．70±0．09）;传统法与化学裂解法、试剂盒法相比差异有统计学意义（P〈0．05）,化学裂解法与试剂盒法相比差异无统计学意义（P〉0．05）,但以试剂盒法为佳。结论3种方法各具优缺点,不同的DNA提取方法会影响结直肠癌患者肠道相关分子微生态的研究结果。与传统法和化学裂解法比较,试剂盒提取法的效率更高,能够检测到更多种类的细菌,更合适肠道相关分子微生态的研究。     

植物基因组DNA提取方法学评析与验证

分子生物学实验多以提取研究对象的基因组DNA作为研究的起点,由于材料的来源、部位、形态等外在性质的不同,以及化学成分、组织结构等内在特点的差异,常会需要在提取基因组DNA时选择不同的方法,或作一些特殊的处理,如有的植物组织含有较多的多糖及酚、酯、萜等次生代谢产物,从而会影响到所提DNA的纯度及随后的实验分析。因而选择一种简单、快速而高效提取基因组DNA的方法常常成为实验中关键的一步。近几年来已有不少关于通用提取方法的报道,另外一些专属性的方法也有了较大进展。本文拟对这些方法加以总结、比较,以期对它们各自的特点作一剖析,并加以实验改进验证,以期能够在今后的实验中更好地应用。     

发菜基因组DNA提取方法比较与分子鉴定研究

目的 以发菜为研究对象比较不同的提取DNA方法,筛选出了一种适合快速提取发菜DNA的方法。方法 分别利用自主研发的硅珠DNA纯化技术、细菌试DNA提取试剂盒和植物DNA提取试剂盒提取发菜基因组DNA。结果 本实验室开发的硅珠DNA提取纯化技术具有快速、无毒以及获得的发菜DNA纯度高等优势。通过PCR扩增和测序得到16S rRNA基因,利用Blast软件进行序列比对,与发菜同源性为99%。结论 本研究研制的发菜基因组DNA提取方法可用于发菜源性的分子鉴定。     

Degradation of transgene DNA in genetically modified herbicide-tolerant rice during food processing

In order to assess the effect of food processing on the degradation of exogenous DNA components in sweet rice wine and rice crackers made from genetically modified (GM) rice (Oryza sativa L.), we developed genomic DNA extraction methods and compared the effect of different food processing procedures on DNA degradation. It was found that the purity, quantity and quality of DNA by alkaline lysis method were higher than by CTAB (cetyltrimethylammonium bromide) method. For sweet rice wine, CAMV35S (cauliflower mosaic virus 35S) promoter and NOS (nopaline synthase) terminator were degraded by the third day, whereas the exogenous gene Bar (bialaphos resistance) remained unaffected. For rice crackers, boiling, drying and microwaving contributed to the initial degradations of DNA. Baking resulted in further degradations, and frying led to the most severe changes. These results indicated that the stability of DNA in GM rice was different under different processing conditions. For sweet rice wine, Bar was most stable, followed by NOS, CAMV35S, and SPS. For rice crackers, CAMV35S was most stable, followed by SPS, NOS, and Bar.     

一种适于ISSR分析的太子参DNA提取方法

目的获得能用于中药太子参简单序列重复区间DNA标记技术等分析研究的高质量DNA。方法以太子参为材料,采用改良CTAB法提取太子参DNA,经紫外分光光度计和琼脂糖凝胶电泳检测。结果改良的CTAB法能获得高质量的太子参DNA,OD260/OD280>1.8,蛋白质、多糖、RNA等去除较彻底,适于简单序列重复区间分析。结论改良的CTAB法所提取太子参DNA适于简单序列重复区间分析。     

Formation of reactive metabolites from benzene

Rat liver mitoplasts were incubated first with [^3H]dGTP, to form DNA labeled in G, and then with [¹⁴C]benzene. The DNA was isolated and upon isopycnic density gradient centrifugation in CsCl yielded a single fraction of DNA labeled with both [^3H] and [¹⁴C]. These data are consistent with the covalent binding of one or more metabolites of benzene to DNA. The DNA was enzymatically hydrolyzed to deoxynucleosides and chromatographed to reveal at least seven deoxyguanosine adducts. Further studies with labeled deoxyadenine revealed one adduct on deoxyadenine. [^3H]Deoxyguanosine was reacted with [¹⁴C]hydroquinone or benzoquinone. The product was characterized using uv, fluorescence, mass and NMR spectroscopy. A proposed structure is described.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday