一种提高PCR产物定向克隆效率的新方法

介绍一种在对PCR产物进行定向克隆之前先用T4DNA聚合酶将PCR产物末端进行补齐,然后进行限制性酶切和克隆,能显著提高克隆效率的新方法。     

两种pUC18高效T载体的构建

T载体是用于直接克隆PCR产物的线性载体.在此之前,克隆PCR片段时一般先用Klenow片段酶或T4DNA聚合酶削平PCR产物两端,克隆过程中又大都不能使用碱性磷酸酶为载体片段脱磷,因为绝大多数PCR引物5’端未磷酸化,T载体的诞生使分子生物学工作者摆脱了这一窘境,而且,T载体的3’端突出的T碱基与PCR产物3’端由于Taq酶非模板依赖的末端转移酶活性而添加的A碱基[1]互补,使载体与PCR产物的连接效率大大提高.由于具有上述优点,T载体从一产生就引起人们极大的兴趣,很多公司也相继推出了各自的T载体系统,并运用该技术改造了很多传统载体.本…     

利用PCR方法鉴定hiTAIL-PCR扩增产物中的质粒骨架片段

T-DNA突变体是研究基因功能的重要资源。高效热不对称交错PCR (hiTAIL-PCR)是克隆突变体中T-DNA插入位点侧翼序列的常用方法。然而我们发现, 利用hiTAIL-PCR克隆到的一些侧翼序列并不对应于宿主的染色体DNA序列, 而是质粒的骨架DNA片段。通过设置1组RB-S4/AC1或者LB-A4/AC1对照反应, 用PCR方法鉴定了hiTAIL-PCR扩增产物中位于T-DNA侧翼的质粒骨架片段。在后续分析中, 通过排除这些片段, 提高了利用hiTAIL-PCR获得宿主染色体DNA片段的效率。同时, 通过调整反应程序, 使得整个PCR的反应时间也大为缩短。在拟南芥(Arabidopsis thaliana) T-DNA突变体drf1侧翼序列的克隆实例中, 对照反应的引入将hiTAIL-PCR中需鉴定的22条扩增产物降至4条, 效率提高了81.8%。     

基于XcmⅠ酶切的高效TA克隆载体的构建

目的：制备基于XcmⅠ酶切的高效TA克隆载体,并检测其克隆PCR产物的效果。方法：设计一对互补配对的寡核苷酸,经过变性及退火后插入质粒pUC19的多克隆位点,从而在该多克隆位点中引入2个XcmⅠ酶切位点,用XcmⅠ酶切后即获得含有3’突出T碱基的T载体;为了提高该T载体的克隆效率,优化了2个XcmⅠ酶切位点之间的碱基数目,排除了载体自连产生白色克隆的可能性,使假阳性大大减少;此外,为了便于完全酶切与未完全酶切载体的分离,在2个XcmⅠ之间插入了一段无关DNA片段。结果：改进得到的T载体可以有效克隆PCR产物,其阳性克隆率可达95％。结论：构建了基于XcmⅠ酶切的TA克隆载体,经过改进的T载体具有很高的克隆效率。     

一种快速高效的基于CcdB致死基因和SmaⅠ酶切位点的克隆体系构建

PCR片段快速准确的构建到克隆载体是分子生物学实验的关键技术之一。快速高效低背景克隆体系的建立可以显著提高PCR产物的克隆效率及大大缩短克隆时间。本实验室开发出一种在常规条件下进行快速高效PCR连接的克隆体系。本研究将含有ccd B致死基因的gateway cassette片段,插入p UC18载体,成功构建p UC18-Ccd B致死载体,在此基础上结合ccd B致死基因内部的SmaⅠ酶切位点,开发出将PCR产物、SmaⅠ限制性内切酶、T4连接酶及p UC18-Ccd B致死载体一起加入离心管后共孵育10 min,即可转化大肠杆菌。对插入片段的重组质粒进行了酶切,PCR和测序验证,证实了该克隆体系高效可行,具有极高的阳性克隆率。该体系的建立具有高效低背景的特点,并且极大的减少了克隆步骤,省去了胶回收及双酶切过程,缩短了克隆时间,同时继承了p UC18载体的优点,具有很强的应用性及推广性。     

PCR产物末端性质的分析研究

利用PCR、UT-PCR、克隆及测序等技术,对强直性肌营养不良基因（MT-PK）3′-非翻译区分别用Taq,Taq＋Pwo DNA聚合酶进行了扩增、克隆和测序,研究了PCR产物末端组成情况,并比较了上述两种DNA聚合酶对PCR产物末端的影响.结果在用Taq DNA聚合酶扩增的PCR产物主要得到3′端突出1个A（占67.3％,35/52）;在Taq＋Pwo DNA聚合酶扩增的PCR产物末端中得到3′端+A的仅占17.4％,而－1的占34.8％,与前者显著不同.表明PCR扩增产物的末端是复杂多样的.     

一种构建新型T载体的简便方法及应用

T载体能够用于PCR产物的快速克隆且易于操作。以常用的克隆载体pGEM-3zf(+)为出发材料,将其进行改造为通用的T载体。通过一步反向PCR方法在该载体中插入带有Xcm I酶切位点的一段序列,在Xcm I酶的切割下产生两端含有T核苷酸的线型核苷酸序列,最终获得用于克隆PCR产物的T载体。外源基因xdh克隆试验表明该载体构建成功,此载体完全可以用于PCR产物的基因克隆试验,为相关载体的改造提供了思路。     

具有除草剂抗性的TA克隆植物表达载体的构建

TA克隆载体由于能够用于PCR产物的快速克隆且操作简单,因此被广泛应用。p CXSN是以TA克隆为连接方式的植物表达载体,T-DNA区筛选标记为潮霉素抗性基因。本实验利用抗除草剂基因bar、gdh A替换载体上的潮霉素基因,构建了两种可以直接连接PCR产物的TA克隆植物表达载体,获得的含有除草剂抗性基因的p CXSN表达载体适合用于基因的正义和反义表达,不需要添加酶切位点,可以直接连接PCR产物,节省载体构建的步骤和时间。     

GM-CSF PCR产物的克隆:一种快速克隆PCR产物方法的建立

由于Taq DNA聚合酶对腺苷A的优先聚合,PCR产物两单链3′端带有一非模板依赖的碱基,所加多余碱基几乎总是A,因此用克隆平端DNA片段的方法克隆这种PCR产物,很难得到阳性重组子。解决上述问题的方法有3种,一是利用T_4 DNA聚合酶的3′外切酶活性,将PCR产物的多余碱基切掉;二是利用3′端带有dT的T-载体进行克隆;另外还可以用Pfu等DNA聚合酶扩增出不带多余碱基的PCR产物,尔后进行平端克隆。我们利用T-载体克隆的原理,酶切、     

一种PCR酶切克隆目的DNA方法

以PCR扩增克隆survivin启动子为例,根据GenBank报道的序列设计引物,PCR扩增克隆survivin启动子。通过软件在线分析扩增的survivin启动子酶切位点,将扩增与预期的片段大小一致的DNA片段进行酶切,再进行测序。结果表明,采取酶切鉴定PCR产物的方法,可以初步对PCR产物是否正确做出判断。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.