高效检测分析玫瑰黄链霉菌Men-myco-93-63线性质粒

【目的】建立一种高效检测和分析玫瑰黄链霉菌Men-myco-93-63内源质粒的方法。【方法】根据“链霉菌线性质粒5′末端都结合有共价蛋白”这一性质,通过改进琼脂糖包埋块制作方法,发明了一项快速高效检测链霉菌质粒种类、构型及大小的技术,称为“包埋块二次处理法”,该方法还能从正反两方面表明共价蛋白的存在与否。【结果】利用此方法高效检测出了玫瑰黄链霉菌Men-myco-93-63中存在两种线性质粒,双向电泳验证了该方法的正确性,推测质粒大小分别约为 47 kb和53 kb。【结论】利用本研究发明的方法首次成功地检测了玫瑰黄链霉菌Men-myco-93-63中的内源线性质粒。     

链霉菌11371基因工程宿主菌的确定

为消除链霉菌11371内源性质粒对pIJ702的限制,以提高pIJ702转化率,确定11371基因工程宿主菌。采用种内接合转移的方法对链霉菌11371衍生菌U3和P2、P5、P7进行内源性质粒的消除。获得接合转移子U3-P7-6,具有转化外源质粒的能力,转化率为17～20个/100个菌,为链霉菌11371转化体系构建、克隆基因结构、功能鉴定和基因定位等研究提供生物材料。     

噬菌体DNA的快速抽提

介绍一种噬菌体DNA的快速抽提方法.用聚乙二醇沉淀噬菌体颗粒,然后经DEAE纤维素纯化处理和酚抽提.与传统的噬菌体DNA纯化方法相比,改进后的方法方便、快速、经济,可获得高纯度的噬菌体DNA.     

濒危植物七子花DNA的提取及分析

用不同的方法抽提濒危植物七子花嫩叶DNA ,利用紫外分光光度法对其质量进行鉴定 ,以紫外分光光度法及琼脂糖凝胶电泳 -溴化乙锭染色法对其含量进行双重测定 ,显示适合七子花DNA抽提的最佳方法是改进的SDS法 ,该法适合RAPD分析。用此法对七子花植株不同器官的DNA进行抽提 ,其含量分别是 :嫩叶 >枝芽 >老叶 >嫩茎 >老茎。     

新生隐球菌基因组DNA不同抽提方法的比较

目的 DNA是进行分子生物学研究的重要基础。在本研究中,我们建立了2种简单快速抽提基因组DNA的方法并可用作PCR扩增的模板。通过比较4种不同的DNA抽提方法以确定哪种更适合进行下一步的基因分析。方法这4种方法是:玻璃珠法,酶法,3%SDS法和氯化苄法。玻璃珠法是用玻璃珠在混漩器上剧烈振荡破碎细胞壁;3%SDS法是将细胞在含10mmol/LDTT的3%SDS溶液中加热,然后用5mmol/LKAc和异丙醇抽提,DNA的产量通过A260测定。结果 3%SDS溶解法、经典酶法、玻璃珠法和氯化苄法的DNA产量分别为0.4154±0.0367、0.8484±0.0756、1.2636±0.2040、0.4070±0.0339(g/L×108CFU/mL)。结论玻璃珠法是最敏感、重复性好、简单、费用合理的抽提方法 。     

海岛棉(Gossypium barbadense)DNA的提纯

介绍一种从棉花(Gossypium barbadense)种子中分离天然状、大分子和高纯度DNA的方法。首先,用有机溶剂(醇、醚和酮)抽提除去脂肪、类脂、棉毒素及其它色素,然后,用胰蛋白酶(Trypsin)和去垢剂(SDS)进行处理脱蛋白;最后,用4％珠状琼脂糖凝胶(Sepharose4B Gel)柱层析分离纯化DNA:此方法简便、有效,一般适用于含大量内源多酚类化合物、色素和类脂植物材料中DNA的抽提和纯化。     

吸水链霉菌应城变种的四个内源质粒及其逐个消除的研究

在改良质粒DNA提取方法的基础上,从三种农用抗生素的同一产生菌——吸水链霉菌应城变种中同时发现四个内源质粒,用双向电泳技术确定了它们均为CCC构型,根据分子量从大到小的顺序将它们分别命名为pHZ1、pHZ2、pHZ3和pHZ4,与已知分子量的CCC质粒分子同步电泳估计它们的分子量依次分别为61kb、4.7kb、4.1kb和3.3kb,基于这四个内源质粒中至少部分个体可能为接合性质粒,可在没有某个质粒的衍生菌株的菌坪上形成“麻点”的假设,我们分离和鉴定了三个质粒逐个消除的10-22衍生菌株,并在光学显微镜下确证了二种类型的麻点。     

GA3对杂交稻“粒间顶端优势”及灌浆期间籽粒内源IAA的影响

以杂交稻协优 413为材料 ,研究了外源GA3 对籽粒内源IAA影响。观察到开花后其优势粒比劣势粒灌浆启动早 ,优势粒灌浆势下降后 ,劣势粒开始灌浆 ,且优势粒灌浆势峰值比劣势粒高 ,这种优劣势粒的灌浆异步现象是一种“粒间顶端优势” ,即劣势粒的灌浆启动迟 ,与优势粒对劣势粒的抑制有关。籽粒灌浆期间优势粒内源IAA含量增加早于劣势粒 ,优势粒内源IAA峰值也高于劣势粒 ;优劣势粒灌浆势变化趋势与优劣势粒内源IAA含量水平的变化趋势相似 ,“粒间顶端优势”似为内源IAA所调节。于初穗时喷施外源GA3,“放大”了单穗中优劣势粒灌浆异步效应 ,优势粒灌浆势更强 ,劣势粒更弱 ,即外源GA3 加强“粒间顶端优势”现象。在试验中还看到初穗期喷施 10～ 40mg/L浓度的GA3 溶液 ,随浓度提高 ,优势粒内源IAA含量增加 ,劣势粒IAA含量更低 ,这种变化似与施用GA3、使优势粒对IAA吸收更多有关     

獐不同组织材料DNA提取的有效方法

根据野外采集的獐肌肉、皮张、毛发、血迹、骨骼和粪便等不同样本的特点采用相适合的DNA提取方法,并对肌肉、皮张、毛发、骨骼的提取进行了改进,通过对线粒体细胞色素b和控制区基因PCR扩增反应以及测序结果证实,这几种DNA抽提方法及相应改进的可靠性,并可以提高野外非损伤性取材在保护遗传学中的应用。     

一种大规模筛选单克隆及提取质粒的改进方法

利用α-互补(X-gal IPTG)从cDNA库中筛选特异克隆,以此去除含有未插入片段和插入片段小的克隆。运用展库的方法,通过辅助噬菌体对库中多个载体的切割,将载体以质粒的形式转化到大肠杆菌中;同时对质粒DNA碱裂解提取方法做了改进,建立了一种简单实用的大量提取质粒DNA的方法。该方法利用特定的蛋白质吸附膜吸附提取过程中的蛋白质,从而去除了使用酚-氯仿抽提蛋白质的复杂过程,减少了质粒DNA的损失,是一次性获得大规模质粒DNA的有效方法。获取的质粒DNA样品在纯度和浓度上都可以满足测序、PCR及Southern杂交等分子生物学实验的要求。     

乳酸菌天然质粒提取方法的优化

将前人报道的乳酸菌质粒提取方法与大肠杆菌质粒提取方法相结合,对常用试剂盒质粒提取方法进行改进,建立了一种快速、安全、高效的乳酸菌质粒提取方法。提取过程中,溶菌酶最佳浓度为20mg/ml,最佳处理时间为30min,同时避免了毒性物质溴化乙锭的使用。多次实验结果表明,采用改进后的方法可明显提高乳酸菌天然质粒的提取效果。且重复性好,为下一步乳酸菌的分子改良奠定了基础。     

A new simple method of curing plasmids in lactic streptococci (Streptococcus cremoris; Streptococcus lactis, plasmids)

A simple method for curing plasmid DNA from lactic streptococci is described. When strains of lactic streptococci are grown overnight at 32 degrees C in an unbuffered medium (M17-) and held at the same temperature for an extended period (96 h), the acid environment induces loss of plasmid DNA of different sizes. If the process of growth in M17- broth followed by extended incubation at 32 degrees C is repeated, most of the plasmids are lost, as revealed by gel electrophoretic profiles of DNA. The method is simple and efficient in curing plasmids of lactic streptococci without use of any mutagenic chemical.     

双磁珠法质粒DNA提取技术及应用

质粒是基因合成与测序领域中使用最为频繁的基因运载工具,然而传统的质粒DNA提取方法面临提取通量低、生产成本高等问题,无法满足日益增长的需求。本研究基于质粒提取原理,开发了双磁珠法(double-magnetic-bead method, DMBM)质粒提取技术,探究了磁珠投入量、质粒DNA片段大小、菌液投入量等因素对质粒提取的影响,并且对比了本技术与商业化质粒DNA提取试剂盒提取DNA质量、提取通量及提取成本。结果表明,双磁珠法质粒DNA提取技术可满足不同细胞密度、不同片段长度的质粒DNA提取。此外,该技术搭载96通道全自动核酸提取仪,提取的质粒DNA纯度更高、提取时间缩短80%、提取成本缩减57.1%,从而实现了质粒DNA提取的高通量、低成本,有效助力基因合成与测序。     

人体蠕形螨的DNA提取与随机引物PCR检测

【目的】探索人体毛囊蠕形螨和皮脂蠕形螨DNA的提取方法。【方法】采用液氮反复冻融研磨法破碎螨体细胞, 选用改良小昆虫DNA提取法、碱裂解法和试剂盒提取法, 分别提取冻存时间在5个月内和8~10个月的毛囊蠕形螨和皮脂蠕形螨基因组DNA, 并用随机引物PCR方法进行检测。【结果】蛋白核酸测定仪检测结果显示, 试剂盒法提取的DNA纯度较高、量较多, 明显优于改良小昆虫法和碱裂解法。随机引物扩增结果显示清晰的DNA指纹图谱, 两种人体蠕形螨DNA指纹具有明显差异。蠕形螨冻存时间影响DNA提取的量, 但对DNA提取的纯度和RAPD指纹图谱影响较小。不同DNA提取方法提取的同一种蠕形螨DNA指纹图谱基本相似, 试剂盒法和改良小昆虫法提取的DNA样本条带多而清晰, 碱裂解法提取的样本条带少而模糊。【结论】液氮反复冻融研磨法破碎蠕形螨细胞是有效的, 蠕形螨冻存时间不宜超过6个月, 试剂盒提取法是提取蠕形螨DNA的好方法。RAPD技术可以用于这两种人体蠕形螨DNA分子水平上的检测和分类。     

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains

Abstract To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.     

Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE

The analysis of microbial communities in environmental samples requires accurate and reproducible methods for extraction of DNA from sample matrices that have different physical and chemical characteristics. Even with the same sample type, variations in laboratory methods can result in different DNA yields. To circumvent this problem, we have developed an easy and inexpensive way to normalize the quantities of DNA that involves the addition of an internal standard prepared from plasmid DNA. The method was evaluated by comparing DNA yields using different DNA extraction procedures, after which the DNA was used for microbial community analysis by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S ribosomal RNA (rRNA) and for quantification of 16S rRNA gene copy numbers in environmental samples by real-time PCR. Our results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples. These methods should also have broad application for various other types of environmental samples.     

番茄加工产品DNA提取方法研究

采用SDS法、CTAB法、热碱法和试剂盒法提取番茄加工产品的DNA,并通过PCR方法是否检测出番茄内源基因来评价DNA提取效果.结果显示,番茄加工产品经弱碱稀释处理后,CTAB法和热碱法提取的DNA利用嵌套PCR检测出内源基因,可以满足基因检测需求.同时对加工产品DNA提取及基因检测需注意的问题进行了探讨.     

德氏乳杆菌保加利亚亚种电转化平台的构建和优化

德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisub sp.bulgaricus)是最具经济价值的乳酸菌之一,在世界上广泛应用于酸奶和其它发酵乳生产。当前对该菌的代谢机制研究甚少。外源基因的转化效率是制约其分子代谢机制研究的重要因素。本研究以pMG36c为材料,对L.delbrueckiisub sp.bulgaricus CH3进行电转化条件研究。结果表明,在电转化过程中,电场强度、质粒的浓度、细胞生长状态均对转化效率有明显影响,得到了该菌株的最适电转化条件为:对数初期的细胞,质粒浓度为100 ng/50μl菌液,在10 kV/cm电场强度下电转化,转化后细胞在复壮培养液中培养3 h后涂布选择性培养基,转化率可达2.6×103CFU/μg DNA。以甘氨酸、醋酸锂、二硫苏糖醇处理细胞壁,发现醋酸锂和二硫苏糖醇共同处理对转化效率有明显改善,可提高转化效率。     

Simple method for extracting plasmid DNA from lactic acid bacteria

Rapid screening and large-scale plasmid DNA isolation procedures are described for lactic acid bacteria, using glass beads to break cells. The rapid screening procedure allows one to obtain plasmid DNA pellets in less than 1 h. This method has been successfully tested on various bacteria from the genera Lactococcus, Leuconostoc, Lactobacillus, Pediococcus, Streptococcus, Enterococcus and Propionibacterium. This procedure yields plasmid DNA with minor chromosomal and plasmid DNA-degraded form contaminations.     

An improved method for rapid purification of covalently closed circular plasmid DNA over a wide size range

An improved method has been developed for the large-scale purification of covalently closed circular (CCC) plasmid DNA molecules of sizes ranging from 4·3 to 73 kb. This protocol uses an alkaline-lysis procedure followed by acid-phenol extraction but with several modifications to previously reported methods. The principal modification is the replacement of NaCl by MgCl₂ in the extraction buffer to improve yield and to remove chromosomal and other non-CCC plasmid DNA. Plasmid DNA can be purified in less than 1 h and used successfully in restriction enzyme analysis and cloning experiments.