改良聚丙烯酰胺凝胶电泳检测DNA

本文介绍了应用聚丙烯酰胺凝胶电泳检测DNA的改进方法。与以往方法相比,本方法从配制凝胶储备液,无需封口的水平灌胶,适当地提高电泳的电压梯度,采用改良银染法检测,省却凝胶固定,银染法DNA的纯化方法,及溴化乙锭染色法的操作等一系列改进措施,使聚丙烯酰胺凝胶电泳检测DNA的操作得到简化,更便捷,并减小了对人与环境的毒害作用。     

突变在基因组进化中的意义

在漫长的进化历史中,各物种间和物种内基因组的差异是如何形成、积累乃至保留下来的,不仅是进化生物学中需要解决的核心问题,也是整个生命科学面临的基本问题之一。对该问题的探求必然要通过对突变的深入了解,因为突变不仅是基因组进化的重要驱动力,还是基因 组进化研究的基础。文章围绕突变的性质及其在基因组进化中的深远意义,系统介绍了国际上相关研究的发展历程,所获得的成果和最新动向。     

单碱基突变的检测

单碱基突变的检测吴学军,柴建华（复旦大学遗传所,上海２００４３３）关键词单碱基突变,检测在临床上有很多遗传病是由基因内的单碱基替换所致。如何快速简便地进行检测,发现这种点突变,是临床医学家的需要。１．单碱基检测方法的回顾用来检测单碱基错配的方法有等位...     

PCR-SSCP分析方法的优化

目的：优化PCR-SSCP分析方法。方法：选择野生型Kir2．3质粒和突变型Kir2．3（2123L）质粒为样本,其PCR结果的鉴定采用2％的琼脂糖凝胶EB显色,SSCP分析采用12％的非变性聚丙烯酰胺凝胶DNA银染显色。PCR产物变性比较了三种方法：即碱变性、SDS变性、直接变性。聚丙烯酰胺凝胶根据丙烯酰胺和甲叉双丙烯酰胺及甘油的比例设计了六个实验组,每组采用两种条件电泳,即：30mA恒流快速电泳和100V恒流过夜电泳。结果：选择直接变性的PCR产物作为变性上样液,并确定了三种凝胶配及该条件下适用的电泳条件。即：丙烯酰胺与甲叉双丙烯酰胺的比例为49比1,凝胶中含1％甘油,4℃,500V高压电泳3min接着30mA恒流快速电泳3h;丙烯酰胺与甲叉双丙烯酰胺的比例为49比1,凝胶中含5％甘油,4℃,500V高压电泳3min接着30mA恒流快速电泳3h;丙烯酰胺与甲叉双丙烯酰胺的比例为29比1,凝胶中不合甘油,4℃,500V高压电泳3min接着100V恒流过夜电泳12h。结论：条件优化的PCR-SSCP是一种筛查基因突变简便、有效的实验方法。     

一种新的制备酸性聚丙烯酰胺凝胶的引发系统

一种新的制备酸性聚丙烯酰胺凝胶的引发系统孙新立＊孙海虹王友爱（河北师范大学生物系,石家庄０５００１６）关键词引发系统;转化率;聚合;酸性凝胶;成胶时间碱性聚丙烯酰胺凝胶电泳已经广泛应用于生化分析的各个领域。但酸性聚丙烯酰胺凝胶电泳、过硫酸铵（ＡＰ）－...     

PCR－SSCP技术在基因点突变检测中的几种策略

ＰＣＲ－ＳＳＣＰ技术在基因点突变检测中的几种策略王吉伟,罗赛群（湖南医科大学分子生物学研究中心,长沙４１００７８）关键词ＰＣＲ－ＳＳＣＰ,点突变检测基因点突变的检测对于分子种群生物学的研究、遗传性疾病预测及分子水平的临床诊断等颇具实用价值。在众多的测...     

黄原胶与丙烯酰胺接枝共聚反应的研究

以过硫酸铵为引发剂,在N2气保护下,研究了黄原胶(XG)与丙烯酰胺(AM)的接枝共聚反应.考察了单体浓度、引发剂浓度、反应温度和反应时间等因素对接枝率及接枝效率的影响,探讨了过硫酸铵引发黄原胶接枝丙烯酰胺共聚反应的基本规律.采用红外光谱(FT-IR)、X射线粉末衍射(XRD)对接枝共聚物的结构进行研究,用热重分析(TGA)法表征了产物的热性能,并初步探讨了接枝机理.     

聚丙烯酰胺板状凝胶的简单干燥方法

我们依据组织逐级脱水的原理,经过摸索得到一种经济简单的凝胶干燥方法,特别是解决了浓度较高(10％—15％)凝胶和3％—25％梯度凝胶的干燥,可将聚丙烯酰胺板状凝胶干燥成一张平整、透明的干胶片,可直接照相或用感光胶片直接印制成负片。现将方法介绍如下:     

聚丙烯酰胺凝胶电泳的快速脱色方法

以牛血清白蛋白为材料进行聚丙烯酰胺凝胶电泳(PAGE)和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),凝胶固定后,用考马斯亮蓝R-250染色后比较传统脱色液(冰乙酸-甲醇溶液)和不同盐溶液(NACL、KCL、CUCL2)的脱色效果的结果表明:PAGE和SDS-PAGE胶,0.25和0.5MOL·L-1NACL,在70℃(PAGE)、50℃(SDS-PAGE)下脱色,约2￣4H,效果好,灵敏度高,背景低。     

一种简易的聚丙烯酰胺凝胶干胶制作法

在聚丙烯酰胺凝胶电泳中经常遇到凝胶干胶制作不好的问题,即使在有干胶机的实验室也常发生凝胶裂开、皱缩、电泳条带扩散、干胶质量不理想的情况,一些文献介绍的方法也常发生类似问题。我们在干胶的方法上作了一些摸索和改进,总结出一种简易的、     

炭疽芽孢杆菌A16R株eag基因缺失突变株构建

【目的】构建炭疽芽孢杆菌A16R株eag基因缺失突变株, 为研究eag基因的功能奠定了基础。【方法】本研究以我国人用炭疽杆菌活疫苗A16R株中eag基因为目的缺失基因,根据炭疽芽孢杆菌Ames株基因组序列,利用软件设计了扩增上下游同源臂以及抗性基因引物,构建了重组质粒,将该重组质粒电击转入炭疽杆菌A16R感受态细胞中,利用同源重组原理筛选到炭疽杆菌A16R株eag基因缺失突变株。在分子水平及蛋白质组学方面对基因缺失突变株进行验证。【结果】成功构建了重组质粒,经同源重组后获得eag基因缺失突变株。PCR鉴定表明目的基因已经丢失;SDS PAGE表明野生株与突变株在93 KDa处有差异蛋白条带,经质谱鉴定分析该条带为目的基因所表达的EA1蛋白;双向电泳结果显示突变株与野生株比较明显缺失3个蛋白点,经质谱分析后确定这3个点都是EA1蛋白。【结论】成功获得炭疽芽孢杆菌A16R株eag基因缺失突变株,为深入研究eag基因的功能奠定了基础,同时也为炭疽芽孢杆菌重要基因功能的研究建立了一个良好的技术平台。     

A novel mutation (4040-4045 nt. delA) in exon 14 of the factor VIII gene causing severe hemophilia A

Hemophilia A is an X-linked congenital bleeding disorder caused by Factor VIII deficiency. Different mutations including point mutations, deletions, insertions and inversions have been reported in the FVIII gene, which cause hemophilia A. In the current study, with the use of conformational sensitive gel electrophoresis (CSGE) analysis, we report a novel 1-nt deletion in the A6 sequence at codons 1328-1330 (4040-4045 nt delA) occurring in exon 14 of the FVIII gene in a seven-year-old Iranian boy with severe hemophilia A. This mutation that causes frameshift and premature stop-codon at 1331 has not previously been reported in the F8 Hemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS) database.     

简单快速的DNA银染和胶保存方法

本文介绍了一套简单快速的DNA银染以及胶保存的方法,整个过程仅需10~15分钟,而且背景浅,条带清楚,灵敏度高,稳定性好。胶保存采用双层玻璃纸夹心法,可长久地保存胶显色时的原貌。以常规PAG胶检测和HLA的SSCP分型为例,利用该套方法进行了银染以及胶的保存,均得到了满意的结果。该方法具有推广价值。Abstract:This paper introduced the simple and rapid methods of silver staining and gel preservation.It was taken only about 10 and 15 minutes to stain a gel.The background of gel was light,the bands were clear,the sensibility was high and the stabilization was well by the method of silver staining.The gel preservation adopted a method named two-layer transparent plastic paper \"Sandwich\" which could keep the gel with primitive colors for a long time.The methods were used on PAG checking and SSCP typing of HLA and the results were satisfactory.The set of methods are expected to be widely used in laboratories.     

单链构象多态性（SSCP）及其在昆虫学中的应用

单链构象多态性（SSCP）能够快速、灵敏地检测基因的点突变,其应用范围日益扩大。本文重点综述了其在昆虫学研究中的作用。bstract:Single-strand conformation polymorphism (SSCP) can detect point mutation quickly and sensitively and its scope of application is enlarged everyday.In this paper,its important roles in entomology are summarized.     

猪雌激素受体基因（ESR）点突变的PCR－SSCP检测

雌激素受体基因(ESR)是控制猪高产仔数的主效基因之一,具有明显的基因效应:BB型比AA型母猪胎总产仔数和产活仔数分别高出1.40～3.37和0.63～3.58头,是目前商品猪育种和生产中重点检测的主要基因之一。常规采用PCR－RFLPs方法区分该基因由点突变造成的3种不同的基因型。本研究建立1种基于PCR的SSCP(single－stranded conformation polymorphism)方法对猪ESR该位置的点突变进行检测,具有操作简便、灵敏度高和不需要酶切等优点,可以在育种实践中广泛应用。Abstract:ESR is a major gene controlling litter size in swine.The sows of BB genotype produce 1.40～3.37 more piglets of the total number born and 1.07～2.40 piglets of the number born alive respectively comparing with those of AA genotype.ESR has been one of the widely detected genes in pig breeding and production.Usually the point mutation of ESR gene was detected by PCR-RFLP approach.The present study established a novel method based on PCR-SSCP,with the advantage of easy maniputation,high sensitivity and no necessity for restriction enzyme digestion.This method may be applied for commercial detection of the point mutation of ESR gene in swine breeding.     

VersusSNP:一种单碱基突变的筛选及分类工具

单碱基突变的筛选和分类是SNP分析的基础。为解决手工进行突变位点挖掘工作的困难,编写了VersusSNP软件。它可以解析并过滤序列比对结果,并根据突变类型将位点加以分类,以图形界面呈现给用户。使用VersusSNP,用户可以直观地了解基因组中单碱基突变的情况。其程序及源代码可以从http://sourceforge.net/projects/versussnp下载。     

Electrophoresis in the presence of Coomassie brilliant blue R-250 stains polyacrylamide gels during protein fractionation

A method of staining polyacrylamide gels in which the dye is electrophoresed together with the sample is proposed. The method cuts short and simplifies the conventional electrophoresis procedure by eliminating the separate poststaining step. In the gels run in the presence of sodium dodecyl sulfate, the method produces protein staining patterns which are quantitatively identical to the ones obtained by conventional staining procedure. Additional advantages of the method are easy control over the degree of staining and homogenous staining independent of the gel thickness and concentration of the dye.     

Effects of N-Terminal Deletion Mutation on Rabbit Muscle Lactate Dehydrogenase

Deletion mutants of rabbit muscle lactate dehydrogenase (LDH) were constructed using polymerase chain reaction (PCR) to study the roles of N-terminal residues. The coding sequences of the first 5 (LD5) and 10 (LD10) amino acids of the N-terminus were deleted and the gene was inserted into the prokaryotic expression vector pET21b. The mutant enzymes were expressed in E. coli BL21/DE3 and were purified. Then their characteristics and stabilities were studied. The results showed LDH was completely inactivated when the first 10 N-terminal amino acid residues were removed, but the mutant (LD10) could have partially restored activity in the presence of structure-making ions. The removal of the first 5 and 10 N-terminal amino acid residues did not affect the aggregation state of the enzyme, that is, LD5 and LD10 were still tetramers. The stabilities of recombinant wild-type LDH (RW-LD), LD5, and LD10 were compared by incubating them at low pH, elevated temperature, and high GuHCl. The results showed that the N-terminal deletion mutants were more sensitive to denaturing environments; they were easily inactivated and unfolded. Their instability increased and their ability to refold decreased with the increased number of amino acid residues removed from the N-terminus of LDH. These results confirm that the N-terminus of LDH plays a crucial role in stabilizing the structure and in maintaining the function of the enzyme.     

Subdivision of equine Tf into H1 and H2

Subdivision of equine TfH into two variants, designated H1 (faster) and H2 (slower), has been accomplished by high voltage, thin layer polyacrylamide gel electrophoresis at pH 7.9. Transferrin H1 and H2 have been shown to be controlled by codominant alleles and gene frequencies of the Tf alleles have been determined in the Australian Thoroughbred, Standardbred. Quarter Horse and Arabian Horse breeds.