Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase

High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.     

Target-oriented and diversity-oriented organic synthesis in drug discovery

Modern drug discovery often involves screening small molecules for their ability to bind to a preselected protein target. Target-oriented syntheses of these small molecules, individually or as collections (focused libraries), can be planned effectively with retrosynthetic analysis. Drug discovery can also involve screening small molecules for their ability to modulate a biological pathway in cells or organisms, without regard for any particular protein target. This process is likely to benefit in the future from an evolving forward analysis of synthetic pathways, used in diversity-oriented synthesis, that leads to structurally complex and diverse small molecules. One goal of diversity-oriented syntheses is to synthesize efficiently a collection of small molecules capable of perturbing any disease-related biological pathway, leading eventually to the identification of therapeutic protein targets capable of being modulated by small molecules. Several synthetic planning principles for diversity-oriented synthesis and their role in the drug discovery process are presented in this review.     

Adaptive recognition by nucleic acid aptamers

Nucleic acid molecules play crucial roles in diverse biological processes including the storage, transport, processing, and expression of the genetic information. Nucleic acid aptamers are selected in vitro from libraries containing random sequences of up to a few hundred nucleotides. Selection is based on the ability to bind ligand molecules with high affinity and specificity. Three-dimensional structures have been determined at high resolution for a number of aptamers in complex with their cognate ligands. Structures of aptamer complexes reveal the key molecular interactions conferring specificity to the aptamer-ligand association, including the precise stacking of flat moieties, specific hydrogen bonding, and molecular shape complementarity. These basic principles of discriminatory molecular interactions in aptamer complexes parallel recognition events central to many cellular processes involving nucleic acids.     

4种水稻基因组DNA微量提取方法对ISSR-PCR试验的影响

[目的]找出适用于ISSR试验的效果好、操作简便的水稻DNA提取方法。[方法]采用高盐十二烷基硫酸钠（SDS）法、十六烷基三甲基溴化铵（CTAB）法及2种型号试剂盒（DP305、DP320）法提取基因组DNA,利用核酸蛋白检测仪及琼脂糖电泳法测定其纯度与浓度,并比较其作为模板用于ISSR-PCR的效果。[结果]发现SDS法与CTAB法提取的DNA浓度与纯度均不高,在ISSR-PCR中虽能扩增出条带,但结果不能重复;试剂盒DP305与DP320提取的DNA浓度与纯度较好,其中试剂盒DP320效果更优,PCR结果能稳定重复。[结论]试剂盒DP320提取DNA的方法是最适合水稻ISSR试验的DNA提取方法。     

Thyrotropin-releasing hormone precursor: characterization in rat brain

To characterize the precursor of mammalian thyrotropin-releasing hormone (TRH), a rat hypothalamic lambda gt11 library was screened with an antiserum directed against a synthetic peptide representing a portion of the rat TRH prohormone. The nucleotide sequence of the immunopositive complementary DNA encoded a protein with a molecular weight of 29,247. This protein contained five copies of the sequence Gln-His-Pro-Gly flanked by paired basic amino acids and could therefore generate five TRH molecules. In addition, potential cleavage sites in the TRH precursor could produce other non-TRH peptides, which may be secreted. In situ hybridization to rat brain sections demonstrated that the pre-proTRH complementary DNA detected neurons concentrated in the parvocellular division of the paraventricular nucleus, the same location as cells detected by immunohistochemistry. These findings indicate that mammalian TRH arises by posttranslational processing of a larger precursor protein. The ability of the TRH prohormone to generate multiple copies of the bioactive peptide may be an important mechanism in the amplification of hormone production.     

含铁蛋白FAO1融合基因的衣藻核转化表达和分析

【目的】fao1编码Candida cloacae中一种含铁辅基的长链脂肪醇氧化酶。通过构建该基因的衣藻核转化表达载体进行转化,根据表达蛋白的生物活性判断fao1能否充分利用衣藻叶绿体中的铁元素行使功能;同时也为需铁固氮酶基因的研究提供方向。【方法】通过PCR把PsaD信号肽、fao1、His-tag标签融合,连接到pDBle载体上;采用玻璃珠转化法,将重组子导入莱茵衣藻（CW15）中;经博来霉素筛选后,获得转基因植株。运用PCR和RT-PCR法检测了转化衣藻,并且用亲和层析柱纯化蛋白,同时进行FAO1活性检测。【结果】重组质粒测序完全正确;PCR和RT-PCR检测转化衣藻基因组DNA和RNA,扩增片段与预期相符;转化衣藻FAO1蛋白纯化洗脱液使ABTS（2,2′-联氨-双（3-乙基苯并噻唑啉-6-磺酸）二胺盐）底物反应变色。【结论】成功构建了信号肽、FAO1和His-tag融合基因的衣藻核转化表达载体,重组质粒已整合到莱茵衣藻基因组中,并成功转录,纯化的蛋白检测有活性。     

环介导等温扩增技术的研究进展

环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术是利用2对特殊设计的引物和一种具有链置换活性的DNA聚合酶,在恒温条件下特异、高效地扩增DNA的新技术,扩增产物为一系列反向重复的靶序列构成的茎环结构和多环花椰菜样结构的DNA片段混合物,电泳图谱呈阶梯式带状分布,反应伴有白色的副产物焦磷酸镁沉淀产生,可通过肉眼定性判断反应结果.LAMP技术已在核酸的科学研究、疾病诊断和动物胚胎性别的鉴定等领域得到了广泛的应用.从LAMP技术的原理、引物组成及反应过程、技术特点、方法延伸和应用等方面对其进行了概述,以期为同类研究提供参考.     

Cloning and expression of a rat brain GABA transporter

A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.     

Carcinogen and microsomal membrane interactions: changes in membrane density and ability to bind nucleic acids

DNA and synthetic copolymer polyribocytidylic-polyriboguanylic acid bind to microsomal membrane. The nucleic acid-membrane complex may be detected by centrifugation in cesium chloride density gradients. The density of the nucleic acid-membrane complex and, in certain cases, the amount of nucleic acid associated with the membrane was changed in the presence of various carcinogenic chemicals.     

Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors

Fragments of exogenous DNA that range in size up to several hundred kilobase pairs have been cloned into yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. Individual clones of yeast and human DNA that have been analyzed by pulsed-field gel electrophoresis appear to represent faithful replicas of the source DNA. The efficiency with which clones can be generated is high enough to allow the construction of comprehensive libraries from the genomes of higher organisms. By offering a tenfold increase in the size of the DNA molecules that can be cloned into a microbial host, this system addresses a major gap in existing experimental methods for analyzing complex DNA sources.     

禽呼肠孤病毒分离株σ2蛋白基因克隆及序列分析

为了解禽呼肠孤病毒（ARV）广西分离株与常用疫苗株在基因水平上的差异,将从广西某鸡场分离获得的2株ARV（R1、R2）分离株的σ2蛋白基因经RT-PCR扩增后,连接pMD18-T载体并转化大肠杆菌DH5α,重组质粒经PCR、双酶切鉴定及序列分析。结果表明,2株ARV分离株的σ2蛋白基因已成功克隆到质粒载体上;经序列分析发现,2株ARV分离株与ARV标准株S1133的核苷酸同源性分别高达99.2%和99.3%,其推导的氨基酸同源性分别高达98.1%和98.4%;两分离株间的核苷酸同源性为99.7%,其推导的氨基酸同源性为98.5%,具有很高的同源性。     

基于链替代扩增-纳米金比色直观检测单链核酸方法的研究

[目的]将链替代扩增技术与核酸修饰纳米金积聚变色的光学特性相结合,设计了一种新型的直观检测3′端暴露单链核酸的方法,实现对单链核酸的高灵敏度检测。[方法]设计一条含有硫代修饰酶切位点的单链核酸（ZDNA）,其酶切位点5′端是能将核酸修饰纳米金变色的Linker序列,3′端是能与Target单链核酸3′端完全互补的H序列。当无Target存在时,Linker会充分暴露,能使核酸修饰纳米金积聚呈现紫色;但是当Target存在时,会与H序列完全互补,作为ZDNA的引物进入链替代扩增循环,形成的新链不断与Linker序列互补,不能使核酸修饰纳米金积聚呈现红色,从而间接检测了Target单链核酸。[结果]通过一系列试验确定检测体系,具体为：40μl修饰纳米金溶液（0.52 nmol/L）加入10μl酶循环体系（ZDNA20 nmol/L,33 mmol/L Tris-HCl（pH值7.9）;10 mmol/L MgCl2;66 mmol/LNaCl;0.5 mmol/LdNTP;0.1 mg/ml BSA;0.05 U/μl klenow;1 U/μl Hinc II）,直观或紫外检测并绘制Target浓度与纳米金积聚程度的标准曲线,表明Target浓度在1~200 pmol/L的范围内呈现良好的线性关系,R2=0.946,最低检测限是1 pmol/L。[结论]链替代扩增-纳米金比色检测单链核酸方法简便、直观、成本低,与传统修饰纳米金比色检测DNA方法相比,灵敏度提高了104倍。     

SELEX技术筛选毒死蜱单链DNA适体

试验旨在利用SELEX技术体外筛选毒死蜱的特异性适体。体外合成全长为91 nt的ssDNA文库,以链亲和素修饰的凝胶为载体、毒死蜱为靶分子进行SELEX(配体指数增强系统进化技术)筛选。利用荧光标记法测定适体的筛选效率、亲和力和特异性,通过MFOLD分析软件对亲和力较高的适体进行二级结构预测和结合位点分析。结果表明,经过15轮筛选后,DNA文库的筛选效率达到44.00%;最终获得9条ssDNA适体,其中适体N23对毒死蜱具有最高的亲和力,其结合活性显著高于N23与水胺硫磷、丙溴磷、氧化乐果的结合活性;二级结构表明茎环结构可能是毒死蜱与适体相互作用的结构基础。     

几种提取乌苏里貉毛囊核酸方法的比较(英文)

[目的]该研究旨在探讨提取乌苏里貉毛囊核酸的可靠方法。[方法]本实验分别利用改进的有机酚法,chelex-100法,PCR缓冲液法和试剂盒法,从乌苏里貉毛发中提取核酸DNA,对所提核酸进行PCR扩增及电泳分析,并且对四种方法进行比较。[结果]经分光光度计测定和凝胶电泳检测,本实验研究结果表明chelex-100提取的核酸有蛋白质等杂质,PCR缓冲液法提取核酸浓度低有机酚法和试剂盒法提取的DNA浓度高,且没有杂带。[结论]本研究为进一步探索高效,简便的提取乌苏里貉毛囊核酸DNA提供了有力的科学理论基础。     

适于转基因产品检测的核酸扩增技术

目前,转基因产品检测主要以核酸为对象,特别是外源重组DNA。靶DNA序列的扩增是转基因产品检测的核心技术,如已经广泛应用的定性、定量PCR技术。随着转基因作物种类的增多、种植范围的扩大,已有方法在某些情况下已不能完全满足检测要求,如高通量多靶标分析。综述了近年来基于核酸水平的检测新技术,并对其未来应用于转基因产品检测进行了分析。     

可用于转基因产品检测中的核酸体外等温扩增技术分析

随着生物技术的快速发展,越来越多的核酸体外扩增技术应用于分子生物学研究领域。根据是否依赖于温度循环,核酸体外扩增技术可分为非等温和等温扩增两大类。核酸体外等温扩增技术由于其有快速、灵敏、不需要特殊仪器等优点,逐渐显示出其广泛应用的潜力。重点介绍了现有的几种核酸体外等温扩增技术的原理、特点及应用,并特别关注这些方法在转基因产品检测中的应用。     

检测禽多杀性巴氏杆菌环介导等温扩增(LAMP)方法的建立

以禽多杀性巴氏杆菌(Pasteurella multocida,Pm)C48-1为研究对象,利用LAMP方法对Pm进行快速检测.结果表明:LAMP方法在恒温65℃下,1 h内就可以检测到Pm;LAMP方法最低可检测100 pg.μL-1Pm基因组DNA.该方法不需要精密的温度循环装置,有恒温加热设备就可以满足检测条件,适合基层临床应用.     

Efficient and nontoxic DNA isolation method for PCR analysis

An optimized method for total DNA extraction from genetically modified sugar beet plants has been presented. Uses of high-toxic organic compounds and high-cost extraction kits for nucleic acid isolation are excluded. A comparative analysis of polymerase chain reaction products was performed with amplification of DNA obtained by two methods.     

猪CFTR基因特异性锌指核酸酶的筛选与鉴定

【目的】获得靶向猪囊性纤维化跨膜传导调节因子(Cystic fibrosis transmembrane conductance regulator,CFTR)基因的特异锌指核酸酶(Zinc finger nuclease,ZFN),为建立CFTR基因敲除猪细胞系提供技术支持。【方法】通过开源式(Oligomerized pool engineering,OPEN)方法筛选,首先以已知三锌指蛋白为框架,随机突变单个锌指关键位点氨基酸编码序列,建立人工三锌指蛋白随机库;然后应用细菌双杂交技术,从库中筛选出能够结合CFTR基因靶位点的三锌指蛋白;最后将获得的锌指蛋白与非限制性核酸内切酶FokⅠ组装成特异ZFN,通过酵母验证体系,检测ZFN靶向切割其识别序列的效率。【结果】获得3个人工三锌指蛋白随机库,每个库约含有2×10⁶个单克隆,通过2轮细菌双杂交筛选,分别获得48个针对CFTR基因左右两侧靶位点的三锌指蛋白。ZFN活性验证结果表明,约90%的ZFN能够在酵母细胞内实现靶向切割,获得了高效特异的ZFN。【结论】获得了猪CFTR基因高效特异的ZFN。     

抗三氟氯氰菊酯截形叶螨遗传因子的PCR研究

通过室内模拟抗性汰选出抗三氟氯氰菊酯截形叶螨种群。利用蛋白酶 Ｋ 及十二烷基硫酸钠、乙二胺四乙酸钠盐等裂解叶螨细胞,提取基因组 Ｄ Ｎ Ａ。参照有关大鼠、果蝇钠通道的核酸序列,设计２５ 、２７ 个核甙酸的引物对,采用聚合酶链反应分别对抗三氟氯氰菊酯种群和敏感种群进行扩增,产物扩增片段为３００ｂｐ 。由扩增结果得知,引物设计合理,并可推测：截形叶螨对菊酯类农药产生强抗药性的原因并非是由于钠通道基因的突变作用引起