cDNA末端快速扩增技术新进展

全长cDNA的获得是基因克隆的重要内容,也是目前人类基因组研究中后 个重要方面,cDNA末端快速扩增（RACE）技术是以PCR为基础,从部分已知的cDNA序列扩增出其他未知部分的5′端和3′端的cDNA序列的一种方法,本文从RACE技术的基本原理方法出发,详细阐述了其存在的缺陷,并对RACE最新研究进展,如Marathon-PCR,Smart-PACE以及“电子”cDNA克隆等作一综述。     

大乳头水螅RACE cDNA文库的构建

目的:为筛选和克隆大乳头水螅发育调控相关基因的全长cDNA,构建大乳头水螅RACE cDNA文库.方法:提取大乳头水螅总RNA后从其中分离mRNA,运用SMART技术构建RACE cDNA文库.为鉴定所构建文库的质量,根据GenBank中大乳头水螅actin基因cDNA序列设计5'RACE和3'RACE的引物及用于扩增actin基因编码区全长序列的引物.结果:琼脂糖凝胶电泳结果表明,RACE cDNA文库中全长cDNA的长度集中在500-2 000bp之间.5'RACE、3'RACE PCR及扩增actin基因编码区全长序列时均以本文构建的大乳头水螅RACE cDNA文库为模板,这3个PCR反应均能扩增出产物,产物大小与目标片段预计大小相似.PCR产物分别经T/A克隆及测序后证明为大乳头水螅actin基因cDNA的相应序列.结论:RACE cDNA文库的成功构建为通过RACE方法获得大乳头水螅功能基因cDNA全长序列奠定了基础.     

中华绒螯蟹卵巢RACB cDNA文库的构建

应用抑制性差减杂交技术,已经获得了中华绒螯蟹卵巢发育过程中差异表达基因的部分cDNA序列。为了进一步获得基因的全长cDNA序列,运用SMART技术,成功构建了中华绒螯蟹卵巢(Ⅲ期)RACE cDNA文库。琼脂糖凝胶电泳结果表明,文库所含全长cDNA的长度主要集中在500—2000bP之间,RACE PCR结果表明,所用基因特异性引物与接头引物皆能扩增出产物,说明所构文库的质量较好,适于用RACE方法从中分离中华绒螯蟹卵巢发育相关基因的全长cDNA。     

一步3’RACE快速构建鸡MnSOD全长cDNA克隆Rapid

本研究尝试将触减 PCR与3’ cDNA末端快速扩增（rapid amplification of cDNA ends,RACE）技术进行结合,仅用一条特异性引物和一条通用引物,成功地实现了从3’末端cDNA库对鸡含锰超氧化物歧化酶（manganese-containing superoxide dismutase,MnSOD）全长cDNA的一步3’RACE快速构建。与常规使用的末端PCR或亚克隆方法相比,该法具有快速、省时、经济和特异性好的优点。Abstract: RACE(rapid amplification of cDNA ends) is a popular technique to rapidly obtain the full-length cDNA. After obtaining the 3’cDNA and 5’cDNA fragments with a overlapped region by 3’RACE and 5’RACE, the full-length cDNA could be generated by end-to-end PCR or subcloning. In this study, 3’RACE combined with touch-down PCR was successfully used for the rapid construction of full-length MnSOD cDNA of chickens. Compared with the conventional end-to-end PCR or subcloning, this method, called one-step 3’RACE, is fast, economical and highly specific. It especially fits the rapid construction of full-length cDNA by RACE method.     

cDNA末端快速扩增技术的研究进展

cDNA末端快速扩增技术是一种基于多聚酶链式反应的技术 ,它的发展大大便利了应用其它方法获得的部分cDNA序列后克隆全长cDNA 5’和 3’末端的工作。不仅RACE方法能在短时间内得到完整的cDNA末端序列 ,而且一些截短的cDNA末端常常也能在RACE的过程中被扩增 ,而这些截短的产物破坏了全长cDNA克隆的获取。许多研究者对RACE的流程提出了改进方案 ,从而提高了该技术的效力。本文介绍了许多已发表的RACE技术关键步骤的改良 ,包括一些具体有效的操作流程 ,如RNA连接酶介导的RACE/连接锚定PCR等 ,还有其有效性的例证。     

SSH和"越轨"RACE分子克隆技术

SSH和“越轨”RACE(step out RACE)均为创建干PCR抑制作用基础上的CDNA基因克隆新技术。SSH具有真阳性率高、不同丰度的mRNA都能被有效分离等优点,“越轨”RACE具有高灵敏度、低背景、操作简便等长处,这两项技术现已经成功应用干大量EST的分离、减数CDNA库的构建、差别表达新基因的分离,以及CDNA全长基因序列的克隆等方面。     

一步PCR快速扩增辽宁碱蓬甜菜碱醛脱氢酶cDNA 3'末端序列

根据已获得的辽宁碱蓬甜菜碱醛脱氢酶cDNA的部分序列,设计一条基因特异性引物,与通用引物并用,一步PCR成功地克隆了辽宁碱蓬甜菜碱醛脱氢酶cDNA 3′末端。与常规的3′RACE法相比,一步PCR法具有快速、简便、经济等优点,是一种非常快捷的扩增cDNA 3′末端序列的方法。 Abstract:Based on part of a known cDNA sequence of Suaeda liaotungensis betaine aldehyde dehydrogenase,we successfully cloned the 3′cDNA end of S.lianotungensis betaine aldehyde dehydrogenase using one step PCR with a gene specific primer and universal primer.Compared with the typical 3′ RACE,one step PCR is rapid,simple and inexpensive.It is very rapid to amplify an unknown cDNA 3′end using this method.     

全长cDNA的获得—RACE及其他方法进展

全长cDNA的获得是基因克隆的重要内容,也是基因组研究的重要内容之一。cDNA末端快速扩增技术（RACE）是获得全长cDNA的主要辅助手段之一,本文对RACE技术的原理、改进和近几年发展起来的新型RACE（包括RNA连接酶介导的RACE、oligo－capping等）作了综述。同时,对用干全长CDNA克隆的其他技术,如引物载体法、固相支持物法作了介绍。     

RACE技术研究进展与展望

近年来,RACE(rapid amplification of cDNA ends)技术,即cDNA末端快速扩增技术,是一种快速扩增cDNA的5′和3′末端的有效方法,在扩增全长cDNA方面得到了广泛的应用。综述了RACE技术的研究进展,总结了优化RACE技术几个关键环节。最后展望了RACE技术的发展。     

悦目金蛛丝腺SMART RACE cDNA文库的构建与鉴定

运用SMART 技术构建了悦目金蛛丝腺SMART RACE cDNA文库.经检测,文库所含全长cDNA的长度主要集中在500 bp～2000 bp 之间;把双链cDNA通过T/A克隆、随机挑选阳性克隆并测序后,得到1条752 bp的全长cDNA序列.以该文库为模板,用依据这条全长cDNA序列设计的基因特异性引物与接头引物进行RACE, 3'RACE 和5'RACE的产物拼接后的全长序列与上述全长cDNA序列一致.结果表明,该文库适于用RACE方法从中分离在悦目金蛛丝腺中表达基因的全长cDNA.本文还对SMART 技术的特点和局限性进行了讨论.     

相异物种同源基因cDNA的快速克隆

介绍了一种结合生物信息学数据库的比较分析,在系统发生相近物种的棱酸保守区设计PCR引物,通过RT-PCR和RACE方法,在相异物种中快速克隆同源基因cDNA的一种简捷方法。     

3' RACE walking along a large cDNA employing tiered suppression PCR

Large genes present particular cloning difficulties, especially when expressed at relatively low levels. We describe a novel method, termed 3' rapid amplification of cDNA ends (RACE) walking, for the rapid determination of unknown 3' flanking sequence of a large cDNA. The technique is a derivative of the anchored PCR 5' RACE procedure but includes a specific and limited second-strand cDNA synthesis and a tiered "panhandle" suppression of nonspecific products. The method generated 900 bp of new sequence for the large tammar wallaby ATRY gene in two easy steps, in which standard 3' RACE and PCR-based cDNA library walking proved unsuccessful. This robust approach represents a new tool for isolating unknown sequence under challenging cloning scenarios such as poor library representation, long coding regions, long 3' untranslated regions, and difficult template regions.     

cDNA cloning by amplification of circularized first strand cDNAs reveals non-IRE-regulated iron-responsive mRNAs

Currently, the rapid amplification of cDNA ends (RACE) is the most common method for PCR cloning of cDNA. Because RACE uses a gene specific primer and one adaptor primer that is shared by all cDNAs may result in numerous nonspecific products that can hinder the cloning process. Here we report a new method that uses circularized first strand cDNA from mRNA and two gene specific primers to amplify both the 5' and 3' cDNA ends in one reaction. A cDNA band of correct size can be obtained on the first pass in this approach. If the correct size is not obtained on the first pass, amplification of cDNA ends can be repeated until the correct size of the cDNA is obtained. We tested this new method on eight mRNAs that we have previously shown to respond to cellular iron levels. We obtained sequences for six mRNAs that were 43 bp to 1324 bp longer than that reported in GenBank and obtained the same length sequence for the other two mRNAs. RNA folding program shows no iron responsive elements (IRE) on these mRNA. In conclusion, our cloning approach offers a more efficient method for cloning full-length cDNA and it may be used to replace the existing method of 5' end cDNA extension. The data enabled us to exclude the possibility that the expression of these iron responsive genes are regulated by IREs.     

cDNA末端快速扩增技术研究进展

全长 cDNA 的获得是基因克隆的重要内容,也是基因组研究中的一个重要方面．cDNA 末端快速扩增技术(RACE)是获得全长 cDNA 的主要辅助手段之一,从 RACE 的原理出发,指出该技术本身存在的优缺点,阐述 RACE 操作中不容忽视的技术要点,对前人对 RACE 的改进加以总结．     

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis

In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3′ end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.