摘要
过去２０年，人们已经分离到了相当数量的与发育相关的基因。这主要借助于蛋白质产物的分离纯化，然后根据其氨基酸结构推算其相应的核苷酸序列，并据此合成寡核苷酸探针，最终从ｃＤＮＡ文库或基因组文库中筛选出目的基因。而未知其编码产物的发育基因的分离克隆则是非常困难的工作，以前广泛应用的主要方法有ＤＮＡ标签法（ＤＮＡｔａｇｇｉｎｇ）［１，２，３］，作图克隆法（ｍａｐ－ｂａｓｅｄｃｌｏｎｉｎｇ）［４］，差别筛选法（ｄｉｆｅｒｅｎｔｉａｌｓｃｒｅｅｎｉｎｇ）［５］和扣除杂交法（ｓｕｂｔｒａｃｔｉｖｅｈｙｂｒｉｄｉｚａｔｉｏｎ）［６，７，８］。这些方法虽然都取得了一定的成功，但由于各自的缺陷性，而限制了它们更加广泛的应用。近年来在ＰＣＲ技术的基础上，人们已经建立了若干种分离克隆植物发育基因的新方法。１９９２年，ＬｉａｎｇＰ和ＰａｒｄｅｅＡ［９］首次提出并运用了ｍＲＮＡ差别显示技术（ｍＲＮＡｄｉｆ－ｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙｒｅｖｅｒｓｅｔｒａｎｓｃｒｉｐｔｉｏｎ－ＰＣＲ，ＤＤＲＴ－ＰＣＲ）来进行基因的分离。实践表明，ｍＲＮＡ差别显示技术在分离、鉴定差别表达的新基因方面与以前的各种技术相比有其独特的优越性，但同时它也存在?
Recently, several new methods about isolating and cloning plant developmental genes have been reported. mRNA differential display is widely used as a speedy and dramatic method, because it only needs very small amount of starting materials and can be used to compare many treatments simultanously. But it has two drawbacks, the high frequency of false positives and the short size of differentially displayed fragments. To overcome these drawbacks, two new methods, called representational difference analysis (RDA) and suppression subtractive hybridization (SSH), are pointed out. In this paper, we discuss in detail the disadvantages of mRNA differential display and the advantages of RDA and SSH. On the other hand, we draw out the progress of these three methods.
出处
《生物工程进展》
CSCD
1998年第2期12-16,共5页
Progress in Biotechnology
关键词
植物发育基因
基因克隆
差别显示技术
mRNA differential display\ Representational difference analysis\ Suppression subtractive hybridization
