利用重叠延伸PCR技术扩增长片段DNA

重叠延伸PCR技术是一种通过寡聚核苷酸链之间重叠的部分互相搭桥、互为模板,通过多次PCR扩增,从而获得目的DNA基因片段的方法.该技术可以高效、快捷、经济地扩增长片段DNA,在基因功能研究方面显示出良好的应用前景.综述了重叠延伸PCR技术的原理、特点以及利用该技术构建长片段DNA的方法和注意事项,并对该技术的应用前景进行了展望.     

亚洲一型口蹄疫病毒YNAs1.1株结构蛋白VP1基因的核苷酸序列分析

提取BHK21细胞增殖的亚洲一型口蹄疫病毒（foot-and-mouth disease virus Serotype Asial)强毒株YNAs1.1的RNA,用一对引物P7,P13经反转录（RT）－PCR法扩增了约674bp的DNA片段。克隆目的基因后,采用双脱氧DNA链末端终止法测得了YNAs1.1的VP1基因36－633核苷酸序列。分析表明,病毒VP1基因的核苷酸序列与以色列以及印度已报道的Asia1型FMDV的同源性分别为82.11%与88.07%,对应的氨基酸序列同源性为87.94％与93.47％。该序列在GeneBank登陆号为AF241566。     

凡纳滨对虾(Litopenaeus vannamei)Hsp70基因PCR扩增及序列分析

目的：为获得凡纳滨对虾的热休克蛋白70（Hsp70）基因并分析其基因序列．方法：根据GenBank中斑节对虾（Penaeus monodon）Hsp70基因的cDNA序列,设计引物,对经高盐法提取的凡纳滨对虾（Litopenaeus vannamei）基因组DNA,采用优化的降落PCR（Touch Downeca）程序,扩增凡纳滨对虾Hsp70基因的全长序列．结果：PCR扩增得到一条长1983bp的目的DNA片段,回收纯化该片段并测定其核酸序列．用DNAman软件分析发现,该核酸序列中不含内含子,编码区全长为1959bp;经BLASTn和BLASTx软件分析发现,该编码区核苷酸序列与斑节对虾、罗氏沼虾（Macrobrachium rosenbergii）的Hsp70基因序列的相似性分别为97％和62．2％．根据核苷酸序列所推导出的Hsp70氨基酸序列,其与斑节对虾、罗氏沼虾的相似性分别为99．9％和92．6％．结论：成功地从凡纳滨对虾基因组DNA中直接扩增出Hsp70基因的全长编码区序列。     

不同倍性鲤科鱼CyclinB基因内含子克隆与进化分析

运用PCR扩增、克隆、测序等技术,分别获得三倍体鲫鲂、四倍体鲫鲂、五倍体鲫鲂和团头鲂、草鱼、鲢鱼、鳙鱼等鲤科鱼CyclinB基因部分DNA序列．结合异源四倍体鲫鲤及其原始亲本红鲫和鲤鱼CyclinB基因对应DNA序列,对不同倍性鲤科鱼类CyclinB基因DNA片段序列进行比较分析．结果表明：由相同引物扩增的染色体数为48的鲂、草、鲢、鳙鱼,只扩增出1条DNA片段,片段长度分别为750bp,950bp,720bp和720bp,而染色体数目加倍的红鲫、鲤鱼、异源四倍体鲫鲤、异源四倍体鲫鲂,扩增出2条DNA片段（1200bp和900bp）,三倍体和五倍体鲫鲂扩增出3条DNA片段（1200bp,900bp和750bp）,每个DNA片段均含CyclinB基因第2,3内含子和第2,3,4外显子．序列分析表明,四倍体鲫鲤、三倍体鲫鲂、四倍体鲫鲂和五倍体鲫鲂与其母本1200bp片段同源性分别为99．5％,98．9％,99．5％和88．7％,与母本900bp片段同源性分别为97．5％,94．6％,94．2％和89．99／6,说明四倍体鲫鲤与多倍体鲫鲂1200bp和900bpCyclinB基因片段与母本红鲫同源性高,在进化上具有偏母性遗传特性．而三倍体和五倍体鲫鲂的750bpCyclinB基因片段-9父本同源性分别高达98．6％和98．2％,说明其来自父本团头鲂．在两性可育的异源四倍体鲫鲤和异源四倍体鲫鲂中存在各自父本CyclinB基因片段序列消除现象．此外,用CyclinB基因内含子3序列构建不同倍性鲤科鱼的系统进化树,结果表明,由CyclinB基因内含子序列构建的系统进化树可以正确反映亲缘关系较近的鲤亚科属间鱼类系统进化关系,而不适合亲缘关系较远的亚科间杂交鱼类系统进化分析．     

中国明对虾与5种虾类线粒体16S rRNA和COI基因片段的序列比较及其系统学初步研究

采用PCR扩增和序列测定等技术,对中国明对虾（Fennerpenaeus chinem如）线粒体DNA16S rRNA和细胞色素氧化酶I（COI）基因片段进行了初步研究,分别得到16S rRNA和COI 2个基因片段的碱基序列,其中16S rRNA基因片段的大小为515bp,碱基A＋T,G＋C的组成分别为66．47％和33．53％;COI基因片段的大小为472bp,碱基A＋T,G＋C的组成分别为62．50％和37．29％．在2种基因片段中,AT的组成明显高于GC的组成,这与果蝇、虾类、蟹类等无脊椎动物的16S rRNA和COI基因片段的研究结果相似．通过对中国明对虾16S rRNA和COI 2个基因片段遗传特征的研究,16S rRNA只有8处核苷酸的碱基在4个群体间表现出差异,COI基因片段中共检测出24个多态性核苷酸位点,其种内变异较低。另外,将本研究所得序列与GenBank中对虾科5个种的16S rRNA基因片段序列进行比较后,发现其聚类关系与传统分类相一致．     

血吸虫膜相关抗原基因的克隆

目的:筛选日本血吸虫(Schistosoma japonicum,S.japonicum)新的疫苗候选抗原基因.方法:以S.japonicum成虫DNA为模板,PCR扩增相关基因,然后将其克隆XpBS-T载体,通过菌落PCR对产物进行检测,对阳性克隆子测序,与GeneBank中的已知相关序列进行比对分析.结果:菌落PCR获得一条与PCR产物一致的DNA片段,序列测定结果表明PCR产物与S.japonicum 22.6 kDa抗原分子核苷酸序列具有高度同源性.其目的基因大小636 bp,具有一个189 bp的ORF,能编码63个氨基酸.ORF不是全长的阅读框,只获得部分编码区.表达产物可含有一个包含9个氨基酸残基的潜在螺旋跨膜片段(25～33位)和一个酪氨酸激酶磷酸化位点(38～39位).讨论:成功克隆出一个与S.japonicum 22.6kDa抗原编码基因有高度同源性的基因.预测该基因为膜相关蛋白基因,可编码血吸虫体表膜相关蛋白.在生理机能上,该膜相关蛋白可能是信号传递分子.     

青海牦牛与云南牦牛乳球蛋白基因5''''调控区核苷酸序列同源性比较

利用一对PCR引物，寡聚核苷酸序列的上游引物为5’-gCg，AAT，TCA，TCC，CAC，gTg，CCT，gC-3’；下游引物为5’-gAg，AAT，TCC，Tgg，ggA，ggg，ACC，TT-3’。经68℃退火及30轮循环的PCR程序后，分别克隆来自我国青海和云南地区牦牛的乳球蛋白基因的5’调控区1447bp的DNA片段。将该片段从琼脂糖凝胶中回收，克隆在pMD1S-T Vector后进行序列测定。序列分析结果表明，青海牦牛该序列的核苷酸序列与文献报道的奶牛该基因的同源性为97．65％，云南牦牛该序列的核苷酸序列与奶牛该基因的同源性为96．8％，青海牦牛与云南牦牛该基因片段的核苷酸序列同源性为99．4％。     

PCR技术在动植物检疫中的应用

一、PCR技术基本原理多聚酶链式反应,英文缩写为PCR,又称无细胞分子克隆法,是近年发展起来的一种快速的特定DNA片段体外扩增法,即一种特异性DNA序列的体外酶促合成方法.PCR利用两个寡聚核苷酸杂交对应链目的区域的侧翼上,一连串反应包括模板变性作用、引物退火和由DNA聚合酶酶促退火、引物延伸的周期性重复产生特定片段的指数积聚.该片段的末端由引物的5′末端决定.因为一个周期所合成的引物延伸产物可作为下一个周期的模板,所以靶DNA拷贝数目在每个周期中近似地呈倍数增加.这样,20个PCR周期可得约百万(2~(20))倍扩增.把扩增产物放进电泳,经溴化乙锭染色后,在紫外灯照射下,肉眼能看见扩增特异区段的DNA带.     

我国不同地域牦牛BLG基因部分核苷酸序列同源性分析

设计一对PCR引物，寡聚核苷酸序列的上游引物为5’-gCg，AAT，TCA，TCC，CAC，gTg，CCT，gC-3’，下游引物为5’-gAg，AAT，TCC，Tgg，ggA，ggg，ACC，TT-3’。经68℃退火及30轮循环的PCR程序后，分别克隆了来自我国甘肃、青海和云南地区牦牛β乳球蛋白基因的5’调控区1447bp的DNA片段。将该片段从琼脂糖凝胶中回收，克隆在pMD18-T Vector后进行序列测定。序列分析结果表明，甘肃牦牛与青海牦牛该序列的核苷酸同源性为98、13％，与云南牦牛该序列的核苷酸同源性为97．65％，青海牦牛与云南牦牛该基因片段的核苷酸序列同源性为99．45％。     

几种鹤性别的分子生物学鉴定

以RAAV01和RAAV02两条随机核苷酸加聚体为引物,通过随机扩增片段多态DNA的方法,在白头鹤,白枕鹤,丹顶鹤等3种4对不同个体中,发现一条约300bp的雌性个性特异带,并应用这一方法成功地鉴别了未知性别的丹顶鹤个体,同时参照已知动物CHD基因序列的设计合成CHD基因引物,采用PCR方法在丹顶鹤雌性个体中扩增出一条206bp的片段,序列分析表明,该序列与已知其他鸟类CHD－W,CHD－Z基因的编码区和内含子区都有较高的同源性。     

Markedness and its Interpretation in Language Use

Language markedness is a common phenomenon in languages, and is reflected from hearing, vision and sense, i.e. the variation in the three aspects such as phonology, morphology and semantics. This paper focuses on the interpretation of markedness in language use following the three perspectives, i.e. pragmatic interpretation, psychological interpretation and cognitive interpretation, with an aim to define the function of markedness.     

Features of Women's language

Language is a means of verbal communication. People use language to communicate with each other. In the society, no two speakers are exactly alike in the way of speaking. Some differences are due to age, gender, statue and personality. Above all, gender is one of the obvious reasons. The writer of this paper tries to describe the features of women＇s language from these perspectives： pronunciation, intonation, diction, subjects, grammar and discourse. From the discussion of the features of women＇s language, more attention should be paid to language use in social context. What＇s more, the linguistic phenomena in a speaking community can be understood more thoroughly.     

An Analysis of Catherine＇s Character——Wuthering Heights

Wuthering Heights, Emily Bronte＇s only novel, was published in December of 1847 under the pseudonym Ellis Bell. The book did not gain immediate success, but it is now thought one of the finest novels in the English language. Catherine is the key character of this masterpiece, because everybody and everything center on her though she had a short life. We can understand this masterpiece better if we know Catherine well.     

Lower Paleozoic oil relationships within Williston Basin, Canada

The Williston Basin is a significant petroleum province, containing oil production zones that include the Middle Cambrian to Lower Ordovician, Upper Ordovician, Middle Devonian, Upper Devonian and Mississippian and within the Jurassic and Cretaceous. The oils of the Williston Basin exhibit a wide range of geochemical characteristics defined as "oil families", although the geochemical signature of the Cambrian Deadwood Formation and Lower Ordovician Winnipeg reservoired oils does not match any "oil family". Despite their close stratigraphic proximity, it is evident that the oils of the Lower Palaeozoic within the Williston Basin are distinct. This suggests the presence of a new "oil family" within the Williston Basin. Diagnostic geochemical signatures occur in the gasoline range chromatograms, within saturate fraction gas chromatograms and biomarker fingerprints. However, some of the established criteria and cross-plots that are currently used to segregate oils into distinct genetic families within the basin do not always meet with success, particularly when applied to the Lower Palaeozoic oils of the Deadwood and Winnipeg Formation.     

低渗透非均质砂岩油藏启动压力梯度研究

理论推导与室内实验相结合,建立了低渗透非均质砂岩油藏启动压力梯度确定方法。首先借助油藏流场与电场相似的原理,推导了非均质砂岩油藏启动压力梯度计算公式。其次基于稳定流实验方法,建立了非均质砂岩油藏启动压力梯度测试方法。结果表明:低渗透非均质砂岩油藏的启动压力梯度确定遵循两个等效原则。平面非均质油藏的启动压力梯度等于各级渗透率段的启动压力梯度关于长度的加权平均;纵向非均质油藏的启动压力梯度等于各渗透率层的启动压力梯度关于渗透率与渗流面积乘积的加权平均。研究成果可用于有效指导低渗透非均质砂岩油藏的合理井距确定,促进该类油藏的高效开发。     

On Tragic Consciousness In The Works By Ernest Hemingway

As an American modern novelist who were famous in the literary world, Hemingway was not a person who always followed the trend but a sharp observer. At the same time, he was a tragedy maestro, he paid great attention on existence, fate and end-result. The dramatis personae's tragedy of his works was an extreme limit by all means tragedy on the meaning of fearless challenge that failed. The beauty of tragedy was not produced on the destruction of life, but now this kind of value was in the impact activity. They performed for the reader about the tragedy on challenging for the limit and the death.     

Design and analysis of generic LBS application developing platform

Location based services is promising due to its novel working style and contents.A software platform is proposed to provide application programs of typical location based services and support new applications developing efficiently. The analysis shows that this scheme is easy implemented, low cost and adapt to all kinds of mobile nework system.     

Achieving Unusual States of Matter Under High Pressure

正The periodicity of the elements and the non-reactivity of the inner-shell electrons are two related principles of chemistry,rooted in the atomic shell structure.Within compounds,Group I elements,for example,invariably assume the+1 oxidation state,and their chemical properties differ completely from those of the p-block elements.These general rules govern our understanding of chemical structures and reactions.Using first principles calcula-     

Exchange-Correlation and Electronic Excitation Energies from Pairing Matrix Fluctuations

We have developed an adiabatic connection to formulate the ground-state exchange-correlation energy in terms of pairing matrix linear fluctuations.This formulation of the exchange-correlation energy opens a new channel for density functional approximations based on the many-body perturbation theory.We illustrate the potential of such approaches with an approximation based on the particle-particle Random Phase Approximation(pp-RPA).This re-     

Density Functional Theory for the Response of Periodic Systems to Electric Fields Based on the Vector Potential Approach

正The electronic and nuclear(structural/vibrational)response of 1D-3D nanoscale systems to electric fields gives rise to a host of optical,mechanical,spectral,etc.properties that are of high theoretical and applied interest.Due to the computational difficulty of treating such large systems it is convenient to model them as infinite and periodic(at least,in first approximation).The fundamental theoretical/computational problem in doing so is that