原子力显微镜观察DNA与内切酶的相互作用

原子力显微镜是观察DNA与内切酶相互作用比较有效的研究技术。实验中,首先把不同种类的内切酶与DNA在含有钙离子的缓冲液中培育30 min,再通过原子力成像观察其相互作用后的复合物形貌。结果表明,二聚体内切酶EcoRV会结合到λ-DNA的单个识别位点,最终形成珠串状结构,而且对DNA有弯曲作用;另一类型四聚体内切酶BspMI除了可以结合λ-DNA的单个位点外,还可以结合2个识别位点而形成圆环状结构。此结果可以直观地观察到两种内切酶与DNA的作用形态,同时对DNA的弯曲与成环等结构给出直接证据。最后给出两种内切酶与DNA相互作用的模型图。     

苯硫脲苦味实验存在问题及重新设计

苯硫脲尝味实验是遗传学实验经典内容,实验内容主要通过收集口腔黏膜上皮细胞,采用50 mmol/L NaOH和1 mol/L Tris (pH8. 0)直接制备DNA模板,选择合适PCR引物扩增出TAS2R38苦味基因,使用内切酶Hae III酶切扩增产物,从而区分出不同基因型。结果表明,该方法解决了上述问题,简化了实验流程,激发了学生学习兴趣,取得了良好的教学效果。     

质粒DNA提取及电泳检测实验改革

为进一步增强学科间的交叉与渗透,提高学生灵活运用多学科知识与方法的能力,对质粒DNA提取及电泳检测实验进行改革。在改进经典碱裂解法提取质粒DNA、琼脂糖凝胶电泳检测质粒DNA的基础上,新增试剂盒方法提取质粒DNA、超微量分光光度计检测质粒DNA以及快速单酶切鉴定质粒DNA等实验内容。改革后的实验不仅促进经典与现代技术、方法的有机结合,而且增强学科间的相互融合,取得了良好的教学效果。     

水稻线粒体DNA的分离纯化及其脉冲电泳分析

为了水稻线粒体基因文库的构建和细胞质雄性不育分子机制的研究,用简化的方法提纯了水稻线粒体DNA,限制性内切酶酶切图谱显示了清晰的带型。HindⅢ酶切产生37条带,Xho酶切产生30条带,该方法重复性好。同时用新技术——脉冲电泳,测定了水稻线粒体DNA的分了量,并就其作为一种新手段,在植物线粒体DNA的异质性、结构和分子大小等方面的研究所具有的优越性进行了讨论。     

DNA分子体外重组实验教学的两种优选方案

DNA分子体外重组技术是基因工程的核心技术之一。国内许多高校在开设这一技术的实验教学中都采用了TA克隆法这一比较落后的实验方法。为了使学生更好地掌握DNA分子体外重组技术,增强技术的实用性,有必要在实验教学内容上作出调整。姊妹PCR克隆法和平末端克隆法是2种简单、经济、重复性好的克隆方法,其中姊妹PCR克隆法不需对目的基因进行酶切处理,即能得到具有各种黏性末端的DNA分子,可直接定向克隆到经相应内切酶消化的载体中。这2种方法不仅能够满足实验教学的需要,也适用于科研工作中各类载体的构建。     

高中生物学教学中涉及的一些重要酶及其作用

1基因工程中的重要酶 1.1限制性核酸内切酶（简称＂限制酶＂）是可以识别DNA的特异序列,并在识别位点或其周围切割双链DNA,使DNA链中磷酸二酯键断开的一类内切酶,被誉为＂分子手术刀＂。     

对现代生物科技专题中一些常见疑问的分析

1限制酶为什么只切割外源DNA而对自身DNA不起作用?限制酶存在于原核生物体内,而原核生物体内限制酶与甲基化酶往往同时存在。甲基化酶同限制性内切酶具有完全相同的识别序列,甲基化酶使识别序列中的某个碱基发生甲基化,从而保护DNA不被限制性内切酶切开。     

“限制性核酸内切酶”考点直击

限制性核酸内切酶主要是微生物体内的一类酶,能将外来的DNA切断.由于这种切割作用是在DNA分子内部进行的,故名限制性核酸内切酶,又称限制酶,是基因工程的"分子     

与核酸有关的几种酶的比较

<正>一、限制性核酸内切酶(限制酶)1.限制酶的来源:在基因工程中,用来切割DNA的工具是限制性核酸内切酶,又称限制酶。迄今为止,基因工程中所用的限制酶绝大部分都是从细菌或霉菌中分离纯化而来的,它们各自可以识别和切断DNA上特定的碱基序列。微生物中限制酶之所以不切断自身DNA,是因为微生物在长期的进化过程中形成了一套完整的防御机制,可以将外源入侵的DNA降解掉。生物在长期演化过程中,含有     

将“人类基因多态性分析”引入遗传学实验课

随着教学改革的深入开展,遗传学实验也在朝着综合性和开放性的方向发展.除了经典的验证性实验外,首都师范大学近几年开设了一些综合性实验项目,让学生运用新的实验技术解决与自身密切相关的遗传学问题.介绍1组利用PCR-RFLP方法进行的人类基因多态性分析实验.学生通过提取自身DNA,然后进行PCR、限制性内切酶酶切及琼脂糖凝胶电泳等一系列步骤,得出自己某个基因(如N-乙酰转移酶2基因)的基因型,再将整个学年学生的数据进行统计分析.通过这组综合性实验,学生掌握了分子生物学基本的实验技术,利用统计学的知识对实验数据加以分析,加深了对理论知识的理解.     

山西省沙棘品种AFLP的初步分析

沙棘体内酚类物质和蛋白质含量较高,影响了基因组DNA提取的产量。采用改良CTAB法对观光木基因组DNA进行提取,经过紫外吸收、琼脂糖凝胶电泳、双酶切和AFLP标记检测,结果表明:改良CTAB法提取的基因组DNA吸光值OD260/OD280为1.7～1.9,DNA无降解现象和杂质污染;基因组DNA双酶切彻底,并且APLP扩增的条带较多而且清晰。从而建立了适合沙棘AFLP分子标记的反应体系,为利用AFLP标记对沙棘进行分子生物学研究及分子育种奠定了基础。     

重组质粒DNA的提取和纯化方法研究

质粒ＤＮＡ的提纯是现代分子遗传学和基因工程的重要技术，本实验对质粒ＤＮＡ的提取和纯化方法进行了比较研究．并通过改进，建立了简便、快速提取和纯化质粒ＤＮＡ的方法．利用此法所提质粒ＤＮＡ收率大，纯度高，能适于细菌转化、细胞转染及酶切分析等进一步实验研究．     

A semester-long project for teaching basic techniques in molecular biology such as restriction fragment length polymorphism analysis to undergraduate and graduate students

Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation. I describe a 12-wk laboratory project suitable for upper-level undergraduates and first-year graduate students, in which the students molecularly locate and map a gene from Drosophila melanogaster called dusky and one of dusky's mutant alleles. The mapping strategy uses restriction fragment length polymorphism analysis; hence, students perform most of the basic techniques of molecular biology (DNA isolation, restriction enzyme digestion and mapping, plasmid vector subcloning, agarose and polyacrylamide gel electrophoresis, DNA labeling, and Southern hybridization) toward the single goal of characterizing dusky and the mutant allele dusky(73). Students work as individuals, pairs, or in groups of up to four students. Some exercises require multitasking and collaboration between groups. Finally, results from everyone in the class are required for the final analysis. Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging. Former students report that the lab was useful in their careers.     

An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonuclease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5'-phosphorylated, circularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.     

以β-淀粉样蛋白为靶的表达载体的构建及鉴定

饱和苯酚氯仿法提取Tg2576转基因鼠基因组DNA,PCR法扩增编码β-淀粉样蛋白的目的基因,利用基因克隆技术构建以β-淀粉样蛋白为靶的表达载体pcDNA3.1-Aβ42×2,应用酶切及测序鉴定表达载体.相应的双酶酶切能够获得插入的目的基因片段(为269bp),测序未发现突变,表达载体构建成功.     

慢性牙周炎患者龈下菌斑的厌氧菌RFLP分型分析

目的：用PCR-RFLP技术,对慢性牙周炎患者龈下菌斑中厌氧菌进行分型.为建立更为合理的分型方法提供依据.方法：选取符合标准的慢性牙周炎患者,提取其龈下菌斑.厌氧条件下分离培养,获得厌氧菌菌株,在血平板培养基培养观察菌落的形态,通过染色观察菌株形态学特征,提取各菌株DNA,利用细菌16sDNA通用引物进行PCR扩增,以限制性内切酶MSP I对分离的22株厌氧菌的PCR扩增产物进行RFLP分型分析.结果：慢性牙周炎患者龈下菌斑中厌氧菌分为二大类.结论：采用PCR-RFLP用于厌氧菌的分型研究较为可靠,对于确定有争议菌种的分类具有重要的意义.     

生物分子系统学研究中的RAPD技术

RAPD技术在Williams等人于1990年首先提出后,在检测DNA多态性方面受到了广泛的应用.RAPD技术以其简单、快速、信息量大等特点渗透在基因诊断,法医检测,生物药品的检验,生物种群关系的研究各个领域.以核酸为指标构建的生物系统进化树,不受主观因素的影响,避免了经典植物系统学的人为影响因素,因而受到广泛应用,在分子系统研究中出现了RFLP,RAPDDNA和RNA测序等分子生物学技术,其中RAPD技术不需要经过DNA分子克隆、分子杂交等繁锁步骤,也不需用放射性同位素显示,并且多态性检出率高,因而受到普遍关注.     

Study on pig growth hormone gene polymorphisms in western meat-type breeds and Chinese local breeds

Chinese Meishan and Jiangquhai pigs are two of the most prolific pigs in the world, but their growth rate is lower than that of Duroc, Landrace and Pietrain pigs. It is suggested that growth rate is regulated by growth hormone. The objective of the current study was to analyze the porcine growth hormone (pGH) gene polymorphisms based on the polymerase chain reaction restriction fragment-length polymorphism method (PCR-RFLP) for three western meat-type breeds (Duroc, Landrace and Pietrain) and two local Chinese pigs (Meishan and Jiangquhai). Five polymorphic restriction sites were detected with the ApaI, MspI, BspI and HhaI restriction enzymes in two amplified fragments (605 bp, -119 to +486; 506 bp, +206 to +711). Breed difference was found only in the 506 bp fragment. There was no difference in allelic frequencies of BspI and HhaI restriction sites among the five breeds (P>0.05). Landrace and Meishan pigs lacked allele G3 of MspI site. The allele G3 frequency of restriction MspI site of the 506 bp fragment in Pietrain pigs was higher than that in Duroc and Jianquhai pigs (P<0.001). For ApaI site, the Meishan pigs lacked allele G1; no difference was found in allelic frequencies among Pietrain, Duroc, Landrace and Jiangquhai pigs (P>0.05). This new and rapid PCR-RFLP typing method is an attractive tool for analysis of porcine growth hormone gene restriction sites. The differences in MspI and ApaI restriction sites may explain the growth difference between the foreign meat-type breeds above mentioned and local Chinese pigs.     

Study on pig growth hormone gene polymorphisms in western meat-type breeds and Chinese local breeds

INTRODUCTIONGrowthhormone (GH)isapeptidehormonewhichregulatesgrowthandvariousmetabolicac tivities (Sterle,1 995;Yuan ,1 996) .InjectionsofGHintogrowingpigsincreasedgrowthrateoftheanimalsandthepercentageofmuscle ,andwhilefataccretionwasdecreased (Bonneau ,…     

Construction of a eukaryotic expression plasmid of Humanin

Objective: To construct a eukaryotic expression plasmid pcDNA3.1 (-)-Humanin. Methods: The recombinant plasmid pGEMEX-1-Humanin was digested with restriction endonucleases BamH I and Hind III and the Humanin gene fragments, about 100 bp length, were obtained. Then the Humanin gene fragments were inserted into eukaryotic expression vector pcDNA3.1 (-) and the recombinant plasmids pcDNA3, l(-)-Humanin were identified by sequencing. Results: Recombinant plasmid DNA successfully produced a band which had the same size as that of the Humanin positive control. The sequence of recombinant plasmids accorded with the Humnain gene sequence. Conclusions: A eukaryotic expression plasmid of Humanin was successfully constructed.