有序差异显示:一种基因表达谱系统比较法

系统研究具有同一基因组的各种细胞群之间基因的差异表达谱十分重要。目前,研究基因差异表达的技术大致有ｍＲＮＡ差异显示［１］、ＲＤＡ［３］、ＳＳＨ［４、５］和ｃＤＮＡ阵列［６］等。近几年,还发展了一些研究基因差异表达谱系统的技术,如ＲＬＣＳ（ｒｅｓｔｒｉｃｔｉｏｎｌａｎｄ－ｍａｒｋｃＤＮＡｓｃａｎｎｉｎｇ）［８］、ＧＥＦ（ｇｅｎｅｅｘｐｒｅｓ－ｓｉｏｎｆｉｎｇｅｒｐｒｉｎｔｉｎｇ）［２］和ＲＮＡ指纹法［９］等。然而,这些技术或较为复杂,或灵敏度偏低。本文拟介绍一种有效的基因表达谱系统比较法——有序差…     

DDRT-PCR技术在研究附植前胚胎基因表达中的应用

甘肃农业大学动物医学院张进隆等5位科研工作者根据哺乳动物附植前胚胎的发育受相应基因的控制。在不同阶段（发育阶段）有不同的差异基因,他们分离并克隆此差异表达基因,以了解胚胎发育的分子机理。他们应用mRNA差异显示技术（DDRT-PCR）的基本原理、特点,即一对细胞的总RNA反转录而成的cDNA模板、PCR高效扩增、     

差异显示的改进及其在木本植物研究中的应用

从差异显示的基本原理出发,概述了差显的优缺点及其近年来在实验方法上的改进,并简要介绍了差显在植物尤其是木本植物研究中的应用。     

mRNA差异显示RT-PCR技术在昆虫学研究中的应用

mRNA差异显示RT- PCR是近 1 0年广泛使用的用于克隆和分析差异表达基因的有效技术。该文简要介绍了差异显示RT -PCR技术的原理、优缺点及其优化方法 ,重点综述了mRNA差异显示RT -PCR技术在昆虫学研究中的应用。     

差异显示技术及其在植物研究中的应用

简要概述了差异显示技术的基本原理和过程及其在植物研究上的应用和进展.     

差异显示技术在橡胶树分子生物学研究中的应用

差异显示技术是从分子水平研究细胞、组织、器官中基因结构和表达变异的手段之一,在巴西橡胶树研究中也已有应用。本文从DNA、RNA和蛋白质水平上介绍了近年来差异显示技术在橡胶树分子生物学研究中的进展,并对其前景作了展望。     

萝卜总RNA提取与mRNA差异显示技术

以萝卜幼小花药与叶片为材料,初步建立了mRNA差异显示技术体系.4种提取总RNA方法中,改进的SDS/酸酚法比CTAB/酸酚法和SDS/碱酚法更合适;改进的热硼酸法(HB)提取的RNA质量也较高,但所需时间较长,成本较高.以改进的SDS/酸酚法获得的RNA进行纯化后,其OD260/OD280在2.0～2.2之间,表明RNA样品杂质较少;总RNA进行反转录与差异显示分析表明,高分辨力的cDNA片段在100～380 bp之间.     

mRNA差异显示技术及其应用

mRNA差异显示技术是国外最近发展起来的一种分离表达水平出现差别的基因的新技术。目前已广泛用于人类及其他动物生长发育基因调控、基因克隆研究,也开始用于植物基因分析、杂种优势机理等方面研究。本文综述这一新技术原理、实验步骤、应用及问题与展望。     

限制性酶切片段差异显示及其应用

差异显示技术(DD)-PCR是一种研究基因表达差异的重要而应用广泛的方法,传统的差异显示法由于在PCR时采用Poly（T）引物和随机引物而导致较高的假阳性率和产物的近Poly(A)非编码区的大量扩增。改进后的限制性酶切片段差异显示技术(RFDD)-PCR采用ToqI酶切双链cDNA,连上特殊设计的接头,再用经特殊设计的特异性配对于接头的引物来扩增,因此能重点扩增编码区并能极大地消除假阳性率。由于扩增时引物就带有荧光或放射性核素标记,还使得差异显示条带的检测更为方便、灵敏。本简要介绍了该法的原理、步骤、应用及其优缺点。     

基因表达中的种群差异

在2月出版的《自然-遗传学》上，研究人员报告。在对不同种族人群的基因测试中发现，基因表达水平上出现的差异性超过25％。     

Global analysis of gene expression by differential display

Differential display (DD) is one of the most commonly used approaches for identifying differentially expressed genes. However, there has been lack of an accurate guidance on how many DD polymerase chain reaction (PCR) primer combinations are needed to display most of the genes expressed in a eukaryotic cell. This study critically evaluated the gene coverage by DD as a function of the number of arbitrary primers, the number of 3′ bases of an arbitrary primer required to completely match an mRNA target sequence, the additional 5′ base match(s) of arbitrary primers in first-strand cDNA recognition, and the length of mRNA tails being analyzed. The resulting new DD mathematical model predicts that 80 to 160 arbitrary 13mers, when used in combinations with 3 one-base anchored oligo-dT primers, would allow any given mRNA within a eukaryotic cell to be detected with a 74% to 93% probability, respectively. The prediction was supported by both computer simulation of the DD process and experimental data from a comprehensive fluorescent DD screening for target genes of tumor-suppressor p53. Thus, this work provides a theoretical foundation upon which global analysis of gene expression by DD can be pursued.     

Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display

We used the differential display technique on total RNAs from roots of Arabidopsis thaliana (L.) Heynh. plants which had or had not been induced for 2 h by nitrate. One isolated cDNA clone, designated Nrt2:1At, was found to code for a putative high-affinity nitrate transporter. Two genomic sequences homologous to Nrt2:1At were found to be localized on the same fragment of chromosome 1 in the Arabidopsis genome. Expression analyses of both low- and high-affinity nitrate transporter genes, respectively Nrt1:1At (previously named Chl1) and Nrt2:1At, were carried out on plants grown under different nitrogen regimes. In this paper, we show that both genes are induced by very low levels of nitrate (50 μM KNO₃). However, stronger induction was observed with Nrt2:1At than with Nrt1:1At. Moreover, these two genes, although both over-expressed in a nitrate-reductase-deficient mutant, were differently regulated when N-sufficient wild-type or mutant plants were transferred to an N-free medium. Indeed, the steady-state amounts of Nrt1:1At mRNA declined whereas the amount of Nrt2:1At mRNA increased, probably reflecting the de-repression of the high-affinity transport system during N-starvation. Received: 4 May 1998 / Accepted: 26 August 1998     

Analysis of differential gene expression in the central nervous system of Schistocerca gregaria by differential display PCR

Within the optic lobes, the mushroom bodies or other parts of the insect brain, information is processed in an area-specific manner. To study the molecular basis of the abilities of the respective areas, the central nervous system of the desert locust Schistocerca gregaria was dissected into different parts, the optic lobes, the “midbrain”, and the thoracic ganglia. Using a simple electrophoretic approach we were able to show area-specific expression of proteins exclusively present in the optic lobes. To study brain area-specific gene expression in more detail, we adapted the differential display polymerase chain reaction to the specific needs of this project. A number of differentially expressed amplicons were identified. The majority of them could be reamplified and their differential expression verified by northern blot analysis. To demonstrate the efficiency of the approach two amplicons with complementary expression patterns were further analysed. Accepted: 31 August 1997     

mRNA差别显示技术及其在生命科学中的应用

ｍＲＮＡ表达水平的变化决定细胞的功能状态,个体发育、细胞增殖分化与凋谢、生理刺激和药物治疗等过程,都会出现ｍＲＮＡ 表达水平的变化。阐明这种变化有助于揭示细胞生理过程的分子机制。该技术无需更多的背景资料,可快速有效地分离表达水平出现差别的基础,这些基因编剧编码的功能多肽在细胞生理过程中扮演着重要角色。     

生物信息学基因表达差异分析

基因的差异化表达由多种因素共同导致,并且与许多疾病的发生和发展有密切联系,对差异化表达的基因进行生物信息学以及生物统计学的分析对于研究细胞调节机制和疾病机理有着重要意义。目前,对差异化表达的基因有以下几种主流的研究方法：DNA微阵列（DNA microarray）,抑制性消减杂交（SSH）,基因表达连续性分析（SAGE）,代表性差异分析（RDA）,以及mRNA差异显示PCR（mRNA DDRT-PCR）。目前许多基因差异化表达数据是建立在时段（time series）基础上,因此对基于时间变化的基因差异化表达分析变得尤为重要。本文将对差异化表达基因的几种主流方法进行详细阐述,并介绍一种基于傅里叶函数的时段基因差异化表达分析。     

Use of bidirectional blots in differential display analysis

We have used bidirectional transfer methods in concert with SMART total cDNA complex probes to sequentially screen differential display arrays. In this report we show the utility of this methodology in examining a manganese superoxide dismutase cDNA fragment which we detected while evaluating the effects of the proinflammatory cytokines IL1-beta, TNF-alpha, and IL6 on human umbilical vein endothelial cell (HUVEC) gene expression. By using parallel hybridization of the bidirectional blots with SMART total cDNA (32)P probes derived from untreated or cytokine-treated HUVECs, differential expression between cell treatments can be clearly evaluated. Subsequent screening using this bidirectional blot method results in detection of modulated cDNA clones. Northern and total cDNA blot hybridization with the cDNA clonal fragment confirmed both modulated expression and the efficacy of this screening method. These procedures allow one to use bidirectional blots to evaluate band modulation on agarose gels which are initially run to evaluate the reamplification of display fragments or to confirm cloned cDNA fragments. Thus, bidirectional blot analysis using SMART total cDNA probes allows direct evaluation of differential display bands from the initial reamplification through plasmid insert cloning, increasing the investigator's ability to eliminate false-positive bands during each step of analysis.     

Differential gene expression during somatic embryogenesis in Coffea arabica L., revealed by RT-PCR differential display

Molecular and biochemical studies of somatic embryogenesis may help to shed light on the mechanisms governing this phenomenon. In this article, a differential display analysis approach was employed to investigate the changes taking place during the induction of somatic embryogenesis in leaf explants and suspension cultures of coffee. Cloned fragments show homologies to several proteins reported in databases, but only one has previously been described as regulated during somatic embryogenesis. By a reverse dot blot modification, the expression pattern of such fragments was evaluated.     

mRNA差异显示

分离差异表达的基因是研究生命调节过程的重要手段,mRNA差异显示技术是一种能成功分离差异表达基因的方法,文章对该方法的基本原理、方法步骤及其应用作了较为详细的介绍。