质料DNA快速提取法

介绍一种质粒ＤＮＡ的快速提取方法，所分离出的ＤＮＡ纯度较邹，全部操作均在一只Ｅｐｐｅｎｄｏｒｆ管中进行，含质粒ＤＮＡ的上清液体中直接点样走琼脂糖凝胶电泳。此法操作简便，整个提取过程只需３０ｍｉｎ便可完成。     

一种快速简单的质粒DNA纯化方法

文中介绍一种快速获取高纯度质粒ＤＮＡ的方法，这种称之为万能微量制备（ＴｈｃＭａｇｉｃＭｉｎｉｐｒｅｐｓ）ＤＮＡ纯化系统在质粒ＤＮＡ纯化过程中不用有机溶剂抽提和乙醇沉淀，而用一万能微柱吸附质粒ＤＮＡ，吸附上的质粒ＤＮＡ可用于水或ＴＥ缓冲液洗脱出来，且不含任何盐或主同分子杂质，纯化的质普ＤＮＡ不需进一步处理即可直接进行ＤＮＡ顺序测定和限制性内切酶消化，整个过程可在１５ｍｉｎ内完成，不失为一种简单可靠的     

绵羊全血中DNA的微量提纯

介绍一种快速，简便地从绵羊冰冻全血中提取高纯度，大分子量ＤＮＡ，并适合边远地区实际室操作的ＤＮＡ提取方法。     

一种上膜虫线粒体DNA提取的新方法

本文根据四膜虫线粒体ＤＮＡ（ｍＤＮＡ）对碱稳定这一特性，建立了一种更为简便的，能直接提取四膜虫线粒体ＤＮＡ的方法。该方法快速简便，重复性好，可用于游离细胞材料ｍｔＤＮＡ的提取。     

以链霉菌高拷贝质粒pIJ101上一个传一性DN…

在链霉菌高拷贝质粒ｐＩＪ１０１上发现并定位了一般０．６ｋｂ的ＤＮＡ片段，这段ＤＮＡ可专一性地刺激不同配对的质业之间共整合体的形成以及在细胞中的累积。形成共整合体的２个质粒之间的亲缘关系可以相近，也可以很远。然而，这段刺激２个质粒分子之间共整合的ＤＮＡ片段在２个质粒上必须处于互反取向，共聚合体才能形成；     

一种简便实用的植物总DNA提取方法

对近年来发展起来的植物基因组ＤＮＡ提取方法进行了改良,提出一种简便,实用的ＤＮＡ提取方法,所得ＤＮＡ的产量和纯度能够满足基因工程操作的要求。     

一种简易的植物核酸提取方法：从干叶和新鲜叶中快速提取…

本介绍了一种植物核酸提取的简易方法，该法只需在常温下接常规的分子生物学操作条件已干燥的和新鲜的植物叶中快速提取总ＲＮＡ和ＤＮＡ。     

青菜花粉管通道法导入外源DNA的研究初报

采用花粉管通道法，将青菜株系７５Ｆ５、１４号大白菜、紫阳等三个品种的总ＤＮＡ及质粒ｐＡｃｔ１－Ｄ ＤＮＡ导入青菜６０５品系，收获青菜种子。种子发芽后分别提取约两周苗龄的小苗总ＤＮＡ，并经ＥｃｏＲⅠ酶切，以ｇｕｓ基因片段为探针做子杂交，证实了外源ｇｕｓ基因已整合到青菜６０５品系中；田间观察还获得了一株与供体大白菜花形相似的变异株。     

质粒PBR322—脂质体的制备及其内含DNA的转移

采用改进的逆相蒸发法（ＲＥＶ）制备卵磷脂／胆固醇（７：３，ｍｏｌ／ｍｏｌ）脂质体，并用于包裹荧光特异标记的质粒ＰＢＲ３２２ＤＮＡ，构建了基因转移载体。然后将纯化的质粒ＰＢＲ３２２ＤＮＡ－脂质体与悬浮的白菜原生质体一起温育。经过ＤＮＡ－ＤＡＰＩ荧光复合物的镜检，证明ＤＮＡ被包裹到卵磷脂／胆固醇脂质体内。被包装的ＤＮＡ能有效地避免外源ＤＮＡａｓｃ的降解作用。且质粒ＤＮＡ通过脂质体与原生质体膜的融合可随     

牛α—s1酪蛋白基因3‘端DNA的PCR扩增,克隆及鉴定

本研究以牛血总ＤＮＡ为模板，用ＰＣＲ技术扩增牛α－ｓ１酪蛋白基因的５＇端３．６ｋｂＤＮＡ片段和３＇端１．５ｋｂＤＮＡ片段的调控区序列，得到了相应的特异性扩增片段．用Ｋｌｅｎｏｗ处理后，将两个扩增片段分别与经Ｓｍａｌ酶切的ｐＵＣ１８质粒载体用Ｔ４ＤＮＡ连接酶连接，转化大肠杆菌ＪＭ１０９．在含有Ｘ－ｇａｌ，ＩＰＴＧ和Ａｍｐ的选择培养基上培养．从白色菌落中提取质粒ＤＮＡ，进行限制酶切鉴定．结果得到一个插入有１．３ｋｂＤＮＡ片段的阳性克隆．对其进行部分序列分析表明，该片段为５＇端缺失约１７０ｂｐ的牛α－ｓ１酪蛋白基因３＇端及下游区序列．     

超顺磁性Fe3O4@SiO2空心亚微球的制备及其在质粒DNA分离纯化中的应用

采用溶剂热法合成超顺磁性空心亚微球,然后通过正硅酸乙酯水解-聚合反应,在亚微球表面包覆SiO₂,形成核壳结构Fe₃O₄@SiO₂空心亚微球。以该Fe₃O₄@SiO₂亚微球为分离介质,实现了大肠杆菌（E. coli）质粒DNA的高效、快速分离。     

用STM证实质粒pUC18DNA分子新构象的存在

用清高妥液法从大肠杆菌ＢＨ１０１细胞制备出的多拷贝质粒ＰＵＣ１８ＤＮＡ分子的构象具有复杂的多样性，我们在对其进行ＳＴＭ研究计，证实了一种不同于已知构象的新构象的存在。     

HBs和HCVc基因双顺反子质粒在BALB/c小鼠体内的分布

 采用注射含双顺反子质粒后,PCR法扩增不同组织中HBsAg和HCVc基因,同时检测HBV和HCV抗体应答水平.研究注射基因免疫用双顺反子质粒在小鼠组织中的分布.pcDNA3.0BApc154S₂S 1次注射BALB/c小鼠后,24 h内主要分布于血液、肝、脾、骨髓、淋巴结、注射部位肌肉、肺和肾等含血液丰富的组织中.注射后2 d主要在骨髓、血液和注射部位肌肉中存在.7 d后仅在注射部位肌肉中能检测出来,并可持续到第9周.组织切片镜检质粒注射初期肌肉细胞轻度浊肿,随后肌膜细胞轻度增生,未见其它明显的组织病理学变化.质粒多次免疫BALB/c小鼠未见明显的临床症状和病理组织学变化.质粒在小鼠体内不同组织存在时间不一致.     

肝细胞靶向性Asor-PLL-DNA复合物的构建

目的:构建肝细胞靶向性Asor-PLL-DNA复合物。方法:将携带外源基因的质粒与脱唾液酸糖蛋白结合在一起,形成一种可溶性的蛋白-核酸复合物。结果:该复合物能通过脱唾液酸糖蛋白受体介导的内吞作用,以非病毒感染的方式,将外源基因导入肝细胞并得以表达。结论:和传统的重组病毒介导的基因转移方式相比,该复合物具有更高的靶向性、安全性,该系统的建立为以肝细胞为靶组织的外源基因介导的肝脏相关疾病的基因治疗提供实验基础。     

Efficient transformation and expression of gfp gene in the edible mushroom Pleurotus nebrodensis

An efficient transformation method mediated by PEG-protoplasts was developed for the newly commercial edible mushroom Pleu-rotus nebrodensis. Two plasmids were used to co-transform protoplasts of P. nebrodensis. One plasmid is pAN7-1 containing a positive selectable marker gene hph conferring hygromycin B resistance. Another plasmid is pBIue-GFP containing a reporter gene gfp conferring green fluorescent protein. PCR and Southern blot analysis showed that hph gene or/and gfp gene were integrated into the genome of P.nebrodensis transformants. The transformation efficiency of the positive selectable marker gene hph was 3 transformants per microgram of plasmid pAN7-1 DNA, which was about 30 times higher than that previously reported in thoroughly studied Pleurotus species such as Pleurotus ostreatus. The transformation efficiency of the reporter gene gfp was 9 transformants per microgram of plasmid pBlu-GFP DNA. The co-transformation efficiency was 23.68%. This is the first report that a "reporter" gene, green fluorescent protein gene can be successfully stably expressed in this Pleurotus species.     

含有肌球蛋白重链启动子的p-EGFP1质粒的构建

从前列腺组织中提取总DNA，以其为模板经PCR扩增得到肌球蛋白启动子，将扩增片段和处理好的p—EGFP1载体相连，连接产物转入到大肠杆菌HB101中，经筛选得到连有myosin启动子的p—EGFP1重组质粒．用酶切和PCR的方法对重组质粒进行鉴定．成功构建的质粒可用于前列腺平滑肌细胞和成纤维细胞的分离，这将对于阐明前列腺基质增生的机理起到重要作用．     

弹性蛋白酶保护因子--Elafin结构基因腺病毒载体穿梭质粒的构建、鉴定及表达

构建能表达弹性蛋白酶保护因子———elafin基因腺病毒表达载体的穿梭质粒,为进一步包装能高效表达结构蛋白的腺病毒载体做准备.以含有elafin基因的真核质粒pEGFP-N1-Elafin为模板,PCR扩增elafin基因,PCR产物以SalⅠ、EcoRⅤ双酶切,定向插入到腺病毒穿梭质粒pAdTrack-CMV中CMV启动子下游SalⅠ与EcoRⅤ位点之间,获得重组腺病毒穿梭质粒pAdTrack-CMV-Elafin,通过SalⅠ及EcoRⅤ双酶切、PCR及插入片段序列测定对该质粒进行鉴定,将pAdTrack-CMV-Elafin转染293细胞,以RT-PCR及Western-Blot检测其在293细胞中的瞬时表达.结果表明:双酶切、PCR及测序鉴定证实,pAdTrack-CMV-Elafin穿梭质粒的插入片段为elafin基因,用pAdTrack-CMV-Elafin穿梭质粒转染293细胞后可见绿色荧光蛋白的表达,RT-PCR和Western-blotting表明其可在293细胞中瞬时表达.     

鸡白痢沙门氏菌直接-多重PCR检测体系的建立

通过对鸡白痢沙门氏菌毒力质粒pSPUV上入侵质粒抗原J蛋白和质粒共转移调控因子的编码基因ipaJ和traJ的序列分析,在特异区域设计PCR引物,经沙门氏菌属不同种及常见肠道致病菌的交叉验证,筛选出三对鸡白痢沙门氏菌特异检测引物ipaJ-417、traJ-387、traJ-476.并且调试出一种利用两种DNA聚合酶的直接三重PCR检测体系:invA-211/traJ-387/16S-495,可以对鸡白痢沙门氏菌进行快速准确的鉴定.     

Isolation of genetic elements that increase frequencies of plasmid recombinants

Some plasmid DNAs, when maintained in wild-type Escherichia coli strains, form high levels of oligomeric species while others remain primarily monomers. One explanation of this observation is that the plasmids that do not form circular oligomers lack a DNA sequence necessary for the formation or maintenance of circular oligomeric species. Here we describe the isolation of segments of DNA from the E. coli genome and other sources that through a recA+ -dependent process: (1) stimulate the conversion of monomeric plasmids to different oligomeric forms, (2) stimulate the conversion of an oligomeric plasmid to a mixture of monomeric and different oligomeric forms, and (3) increase the frequency of recovery of figure-8 molecules. Both cis-acting and trans-acting elements were found. These elements seen to act by stimulating either the frequency of the recombination events that lead to the interconversion of different oligomeric plasmid DNA molecules or some process involved in the maintenance of newly-formed recombinant molecules.     

Giant linear plasmids in Streptomyces which code for antibiotic biosynthesis genes

A number of examples of circular plasmids with specific functions are known in both prokaryotes and eukaryotes. Several linear plasmids have also been identified, but these are all relatively small: large linear plasmids cannot be separated from chromosomal DNA by conventional techniques. There are several cases where the genetic evidence suggests that a character is encoded by a plasmid but no plasmid can be physically detected. This has been the case for antibiotic synthesis genes in Streptomyces; in particular a plasmid SCP1 in Streptomyces coelicolor has been shown to be involved in methylenomycin production by genetic evidence. We report here the application of orthogonal-field-alternation gel electrophoresis to the isolation of linear plasmids from Streptomyces. We have discovered a large linear plasmid of around 520 kilobases in Streptomyces lasaliensis and subsequently similar giant linear plasmids in other Streptomyces strains. We have confirmed that genes for methylenomycin biosynthesis are located on a series of giant linear plasmids in S. coelicolor. These observations may bear on the genetic variability and unstable genetic character of Streptomyces species.