共查询到18条相似文献,搜索用时 156 毫秒
1.
2.
3.
细胞在增殖,分化及对外界刺激反应过程中,可伴随某些特殊基因的表达,利用这一特性,通过比较细胞在不同状态及不同分化阶段基因表达的差异,可以发现与细胞分化/生长相关的基因,近年来国外学者发展了几种能有效进行差异表达基因分析的技术和方法,包括差异显示PCR,代表性差异分析,抑制性消减杂交及DNA微阵列等。本文主要对目前主要技术方法作一综述。 相似文献
4.
运用多种策略改良差异显示PCR 总被引:1,自引:0,他引:1
目的:改进差异显示PCR技术,提高其在筛选差异表达基因方面的效率。方法:①采用单碱基金铆钉引物;②增加引物长度;③提高PCR反应的严谨性;④应用同步重复测序电泳。结果:减少经典方法的工作量,降低了非特异性和假阳性率。用改良技术研究热适应大鼠下丘脑基因的差异表达,发现了一个差异表达基因片段,斑点杂交证实为阳性片段。结论:改进后的差异显示PCR技术是一种研究基因差异表达从而发现新基因或基因新功能的有效方法。 相似文献
5.
用DDRT-PCR方法克隆小鼠精子发生早期相关基因的EST 总被引:1,自引:0,他引:1
为了避免差异显示技术中的放射性污染 ,并用之于筛选、克隆精子发生早期的相关基因 ,分离纯化了小鼠的原始精原细胞及B型精原细胞 ,提取其总RNA ,逆转录获得cDNA ,以荧光差异显示方法筛选差异表达基因。利用斑点杂交技术对差异片段进行快速鉴定以排除假阳性。选取 16条差异显著的片段做克隆测序 ,通过Gen Bank/Blast比较 ,有 7个片段属于新的EST ,且均表现为在B型精原细胞中表达强度高于原始精原细胞。提交Gen Bank获得注册号。从中选取较有意义的 3条基因片段通过半定量RT PCR方法进一步验证其表达特征。和传统的差异显示方法比较 ,文中所采用的mRNA差异显示技术可快速排除假阳性结果 ,避免同位素标记带来的放射性污染。结果表明所获得的 7个新EST均表现为B型精原细胞中高表达 ,这些表达升高的基因可能与其后精子发生过程中的一系列特殊现象 (如减数分裂、变态成形 )有关 ,为生精细胞的分化做物质上的准备。 相似文献
6.
7.
8.
9.
10.
差异表达基因分离技术的研究进展 总被引:1,自引:0,他引:1
分离并克隆差异表达基因是生命科学的研究热点.近年来,以差示筛选、扣除杂交等基本方法为基础,先后出现了抑制差减杂交,微阵列技术等多种分析差异表达基因的技术, 使差异表达基因分离方法不断完善.对这些方法的优缺点、发展趋势及应用前景进行了简要综述. 相似文献
11.
The nonradioisotopic representation of differentially expressed mRNA by a combination of RNA fingerprinting and differential display 总被引:5,自引:0,他引:5
Norbert Kociok Klaus Unfried Peter Esser Ralf Krott Ulrich Schraermeyer Klaus Heimann 《Molecular biotechnology》1998,9(1):25-33
In many applications, an understanding of differentially expressed genes in different tissues, or owing to an applied stimulus
is important. However, the wide use of two rather similar polymerase chain reaction (PCR)-based techniques for the identification
of differentially expressed mRNAs (RNA fingerprinting by arbitrarily primed PCR [RAP-PCR] and differential, display [DDR-PCR]
has shown, that reproducibility is still a problem. By combining features of both RAP-PCR and DDRT-PCR a technique has recently
been developed that avoids some of the disadvantages, but the use of radioisotopes for band detection still limits its application.
We have improved this technique for analyzing differentially expressed mRNA by resolving the amplified products on nondenaturing
polyacrylamide gels and subsequently staining the gels with silver nitrate. Our modification allows the identification of
differentially expressed bands with a very high accuracy. Therefore these bands can be very easily reamplified and sequenced
directly. Subsequently the differential expression can be verified by semiquantitative RT-PCR with specific primers derived
from sequence data. These improvements, together with nonradioactive sequencing techniques, make it possible to do DD analysis
completely without a health hazardous owing to radioactivity. The nonradioisotopic differentially expressed mRNA-PCR (DEmRNA-PCR)
is a reliable and useful modification of available differential expression methods. 相似文献
12.
筛选差异表达基因和蛋白质的方法进展 总被引:10,自引:1,他引:9
分离和鉴定差异表达基因和蛋白质不仅有助于发现基因和蛋白质的功能,更有助于揭示某些疾病的发生机理.目前筛选差异表达基因的方法主要有差异显示PCR方法(differential display RT-PCR,DDRT-PCR)、消减杂交法(subtractive hybridization,SH)、基因芯片技术(DNA chip technique)和基因表达的系统分析(serial analysis of gene expression,SAGE)等,其中消减杂交法中又先后建立了代表性差异分析技术(representational difference analysis,RDA)、抑制消减杂交法(suppression subtractive hybridization,SSH)和获得全长基因的消减杂交法(full-length-gene-obtainable subtractive hybridization).筛选差异表达蛋白质的方法主要有双向电泳技术(two-dimentional gel electrophoresis)和噬菌体全套抗体库技术(phage display antibody repertoire library technique).这些方法各有特点,各有利弊,研究者可根据自己的需要选择适合于自己的方法. 相似文献
13.
14.
Regard Stephania C. Egeland Daniel B. Tannoch Vivien J. Claycomb William C. 《Molecular and cellular biochemistry》1997,172(1-2):111-120
An estimated 15,000 different mRNA species are expressed in a typical mammalian cell. The differential expression of mRNAs in both a temporal and cell-specific manner determines the fate of the cell and creates the organism. Analysis of this differential gene expression has become a central aim of many laboratories attempting to understand the mechanisms underlying various biological processes. Currently, we are using a technique called differential display to analyze the differential expression of genes in cardiomyocytes. Differential display is a rapid and powerful technique that was introduced by Liang and Pardee in 1992. Since that time, it has been successfully applied by several groups, and it is quickly becoming a standard method for studying differential gene expression. Here, we present a detailed article discussing the differential display methodology and how we have utilized it to identify potential genes involved in cardiomyocyte proliferation. Furthermore, we have provided a list of materials and supplied examples of data obtained, in an effort to allow the reader to perform the technique with success in their own laboratory. 相似文献
15.
Differential display (DD) is a novel PCR-based technique, very commonly used to study differentially expressed genes at the
mRNA level. In this paper we report a modified version of this technique that we have used to study the differences between
the mRNA population from brain tissue of adult and old rats. We have modified the technique to enhance reproducibility and
reduce false positives and redundancy. It is fast and does not require any expensive or uncommon reagent. We choose to call
it as subtractive differential display as it is a differential display performed over subtracted mRNA population. We have
used this protocol successfully to clone a number of age-related differentially expressed sequences from rat brain that need
to be sequenced to establish the gene identity. 相似文献
16.
17.
18.
降低mRNA差异显示技术假阳性率的一种方法 总被引:17,自引:0,他引:17
为了探讨降低mRNA差异显示技术假阳性率的方法 ,进一步提高此技术的可靠性 ,提取了手术切除肝癌及非癌肝组织成对标本的总RNA ,逆转录获得cDNA片段 ,以mRNA差异显示方法筛选差异表达基因 ,选取较明显的一条差异表达条带 ,行进一步PCR扩增 .分别对PCR产物及其经TA克隆后随机挑选的 6个单克隆质粒DNA进行序列分析 ,并通过GenBank BLAST数据库进行序列的同源性比较 ,以Northern杂交予以来源确认 .自 72 0余条扩增条带中共选出 2 8条差异条带 .序列分析及同源性比较表明 ,所选择条带的PCR产物为一可能的新基因片段 ;而随机选择的 6个TA克隆质粒DNA中 ,有 4个为同一已知基因片段 ,一个为另一已知基因片段 ,一个为一可能的新基因片段 .同源性比较表明 ,PCR产物直接测序所得序列与TA克隆质粒DNA的 6个片段不具同源性 .结果表明 ,mRNA差异显示条带可能由 1条以上分子量相似的片段构成 ,直接对PCR产物行序列分析并以其为探针进行Northern杂交 ,是导致出现假阳性片段的原因之一 .将PCR产物进行TA克隆 ,对单克隆质粒DNA进行序列分析并以其为探针进行Northern杂交 ,可能是解决此问题的一种较好方法 . 相似文献