酵母菌中质粒DNA的快速检测

随着基因工程研究的发展,近年来酵母质粒作为基因工程载体的研究已获得明显进展。因此,开展酵母质粒DNA快速检测方法的研究,将是很有意义的。目前质粒在啤酒酵母(Saccharomyces cerevisiae)中研究较多。1982年龚启蕙等报道了啤酒酵母7209—1A及1984     

植物线粒体DNA质粒研究进展

本文列出了已发现的高等植物中的线粒体DNA质粒,按分子形状分为线粒体环状DNA质粒和线粒体线状DNA质粒,环状线粒体DNA质粒的特征是分子较小, 序列中有正向/反向重复序列,ORF一般较小。线状线粒体DNA质粒的特征是分子较大,末端有重复序列,5'端与蛋白质共价结合,有较长的ORF。还分别介绍了它们的复制机制、转录和起源。质粒间及质粒与核基因组、线粒体基因组、叶绿体基因组的同源性也作了介绍。最后,综述了植物线粒体DNA质粒与植物的细胞质雄性不育（CMS）之间的关系。     

质粒DNA抗原芯片检测抗双链DNA抗体的初步研究

目的:建立以质粒DNA作为抗原的检测血清中抗双链DNA(dsDNA)抗体的芯片方法,并与酶联免疫吸附实验比较,初步探讨用芯片法检测抗dsDNA抗体的临床价值。方法:将原核表达载体质粒pcDNAⅡ用质粒DNA快速抽提试剂盒提取纯化DNA后按1∶2稀释,用点样仪点在经3-氨丙基三乙氧基硅烷(APES)修饰的玻片上,温孵后用含有1%小牛血清白蛋白和2.5%蔗糖的PBST封闭,以Cy3标记的人IgG为二抗,建立检测dsDNA抗体的芯片方法,并与德国欧蒙公司生产的抗双链DNA检测ELISA试剂盒做比较,对包括58例系统性红斑狼疮(SLE)、25例干燥综合征(SS)、10例皮肌炎(DM)和7例类风湿关节炎(RA)在内的病人和60例健康人对照进行了抗dsDNA的对比检测。结果:对阳性标本的检测,与现用常规检测方法ELISA相比,芯片检测抗dsDNA的灵敏度为91.3%,特异度为90.7%,阳性预测值为89.3%,阴性预测值为92.5%;对健康对照的检测,2种方法均为阴性,符合率为100%。结论:与ELISA相比,用质粒DNA作为抗原建立的芯片方法的灵敏度和特异度较高,为今后建立同时检测多个自身抗体的芯片奠定了基础。     

较长单链DNA在质粒DNA中定位插入

一般重组DNA的方法都是用T_4-DNA连接酶把限制性内切酶处理过的外源DNA片段和载体DNA,连接到一起,但这种方法必须要有合适的内切酶切点,因而使得这种方法的灵活应用受到一的定限制。     

棒状杆菌中质粒DNA的快速检测方法

在棒状杆菌质粒DNA快速检测中,比较了三种方法:强碱法、快裂法和我们设计的酶法。用强碱法抽提的质粒带很淡,快裂法其次,且不能区别两个分子量相近的质粒;而用酶法则显带清晰,且能很好地区分两个大小相近的质粒,分辨率高,认为在棒状杆菌质粒快速检测中是一种很好的方法。     

碱裂解法提取质粒DNA的研究

对质粒DNA及其结构、形态和功能进行了综述 ,介绍了碱裂解法提取质粒DNA的基本步骤 ,并对其提取过程中的注意事项进行了剖析 ,分析比较了不同提取方法的优点     

一种提取质粒DNA的改良方法

本文详细介绍了一种改良碱裂解法提取质粒ＤＮＡ的方法,该法采用ＮＨ４Ａｃ代替苯酚和氯份的抽提过程,得率高,质量好,完全达到了分子生物学常规实验的要求,如酶切、连接、转化大肠杆菌、ＰＣＲ等,甚至用于序列测定和植物遗传转化,该法重复性好,操作简单、实用．     

DNA疫苗的质粒型细胞因子佐剂

细胞因子是机体产生的一系列免疫效应和免疫调节蛋白。在近几年,质粒型细胞因子作为能增强DNA疫苗免疫应答的佐剂已引起了研究者广泛的关注。本文就细胞因子的生物学机制、T细胞分化和利用质粒型细胞因子佐剂优化DNA疫苗等方面的进展进行了讨论。     

大肠杆菌质粒DNA提取方法的优化

目的:简化操作流程,缩短提取时间,降低试验对操作者的危害。方法:该方法去除酚、氯仿等有害试剂,采用LiCl沉淀去除质粒制备物中小片段核酸(包括DNA和RNA);工程菌生长至对数期时通过加入氯霉素后不仅方便质粒DNA的提取过程中蛋白质的去除,而且可使质粒的拷贝数进一步增加,提高质粒DNA产量的目的。结果:实验改进方法所提取的质粒DNA产量高于常规方法,达到20μg/mL。结论:改进方法提取的质粒DNA,其下游的内切酶消化,PCR、重组质粒鉴定、转化大肠杆菌等实验的结果和重复性都令人满意,完全可用于一般的分子生物学研究。     

Molecularly imprinted cryogel for L-glutamic acid separation

A molecular recognition based L-glutamic acid (L-GLU) imprinted cryogel was prepared for L-GLU separation via chromatographic applications. The novel functional monomer N-methacryloyl-(L)-glutamic acid-Fe(3+) (MAGA-Fe(3+) ) was synthesized to be complex with L-GLU. The L-GLU imprinted cryogel was prepared by free radical polymerization under semifrozen conditions in the presence of a monomer-template complex MAGA-Fe(3+) -L-GLU. The binding mechanism of MAGA-Fe(3+) and L-GLU was characterized by Fourier transform infrared (FTIR) spectroscopy in detail. FTIR analyses on the synthesized MAGA-Fe(3+) -GLU complex reveals bridging bidentate and monodentate binding modes of Fe(3+) in complex with the carboxylate groups of the glutamate residues. The template L-GLU could be reversibly detached from the cryogel to form the template cavities using a 100 mM solution of HNO(3) . The amount of adsorbed L-GLU was detected using the phenyl isothiocyanate method. The L-GLU adsorption capacity of the cryogel decreased drastically from 11.3 to 6.4 μmol g(-1) as the flow rate increased from 0.5 to 4.0 mL min(-1) . The adsorption onto the L-GLU imprinted cryogel was highly pH dependent due to electrostatic interaction between the L-GLU and MAGA-Fe(3+) . The PHEMAGA-Fe(3+) -GLU cryogel exhibited high selectivity to the corresponding guest amino acids (i.e., D-GLU, L-ASN, L-GLN, L-, and D-ASP). Finally, the L-GLU imprinted cryogel was recovered and reused many times, with no significant decrease in their adsorption capacities.     

Anion exchange purification of plasmid DNA using expanded bed adsorption

Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH=8.0) buffer at an upward flow of 300 cmh^-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh^-1. Purification factors of 36±1 fold, 26±0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed–values of 35±2 and 5±0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.     

Fractional precipitation of plasmid DNA from lysate by CTAB

Preparative-scale purification of plasmid DNA has been attempted by diverse methods, including precipitation with solvents, salts, and detergents and chromatography with ion-exchange, reversed-phase, and size-exclusion columns. Chromatographic methods such as hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), and size exclusion chromatography (SEC) are the only effective means of eliminating the closely related relaxed and denatured forms of plasmid as well as endotoxin to acceptable levels. However, the anticipated costs of manufacturing-scale chromatography are high due to (a) large projected volumes of the high-dosage therapeutic molecule and (b) restricted loading of the large plasmid molecule in the pores of expensive resins. As an alternative to chromatography, we show herein that precipitation with the cationic detergent, cetyltrimethylammonium bromide (CTAB), is effective for selective precipitation of plasmid DNA from proteins, RNA, and endotoxin. Moreover, CTAB affords novel selectivity by removal of host genomic DNA and even the more closely related relaxed and denatured forms of plasmid as earlier, separate fractions. Finally, plasmid that has been precipitated by CTAB can be purified by selectively dissolving under conditions of controlled salt concentration. The selectivity mechanism is most likely based upon conformational differences among the several forms of DNA. As such, CTAB precipitation provides an ideal nonchromatographic capture step for the manufacture of plasmid DNA.     

Separation of genomic DNA, RNA, and open circular plasmid DNA from supercoiled plasmid DNA by combining denaturation, selective renaturation and aqueous two-phase extraction

In the current study we developed a process for the capture of pDNA exploiting the ability of aqueous two-phase systems to differentiate between different forms of DNA. In these systems scpDNA exhibits a near quantitative partitioning in the salt-rich bottom phase. The successive recovery from the salt rich bottom phase is accomplished by a novel membrane step. The polish operation to meet final purity demands is again based on a system exploiting a combination of the denaturation of the nucleic acids present, specific renaturation of scpDNA, and an ATP system able to differentiate between the renatured scpDNA and the denatured contaminants such as ocpDNA and genomic host DNA. This polish step thus allows a rapid and efficient separation of scpDNA from contaminating nucleic acids which up to date otherwise only can be accomplished with much more cumbersome chromatographic methods. In a benchmark comparison, it could be shown that the newly developed process exhibits a comparable yield to an industrial standard process while at the same time showing superior performance in terms of purity and process time. Additionally it could be shown that the developed polish procedure can be applied as a standalone module to support already existing processes.     

Affinity binding of cells to cryogel adsorbents with immobilized specific ligands: effect of ligand coupling and matrix architecture

The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.     

Quantitation of plasmid DNA in aqueous two-phase systems by fluorescence analysis

The development of aqueous two-phase systems for plasmid purification from Escherichia coli cell lysates requires a reliable DNA quantitation method. Plasmid DNA was quantified by fluorescence using PicoGreen nucleic acid stain. Linearity was obtained up to 40 ng plasmid ml^–1. Two polyethyleneglycol (PEG)/salt systems were studied, PEG 600/K₂HPO₄ and PEG 300/K₂HPO₄. The average plasmid recovery was 41% in the bottom phase of the first system and 35% in the top phase of the second system. This method has proved to be simple and reproducible.     

Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.     

大规模生产制备质粒DNA技术进展

质粒DNA疫苗或基因治疗剂是生物制品的新品种。鉴于质粒DNA表达效率低、持续时间短,需建立一套大规模生产制备质粒DNA的工艺,即发酵、碱性裂解、分离纯化及质量控制。目前质粒DNA大规模生产已经达到千克级水平,一些产品正在进行临床实验。     

Rapid plasmid DNA isolation from Lactococcus lactis using overnight cultures

A simple and quick method has been developed to isolate plasmid DNA from Lactococcus lactis using overnight or stationary-phase cultures which therefore eliminates the need for subculturing for generating log-phase cultures that are necessary with existing methods. The new method was effective in isolating plasmids, from 1.4 to 64 kb, from the three subspecies of Lactococcus lactis. The resultant DNA was of high yield and purity and therefore no additional purification steps were required for down-stream molecular procedures.