Identification and characterization of "zinc-finger" domains by the polymerase chain reaction.
Identification and characterization of "zinc-finger" domains by the polymerase chain reaction.
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通过聚合酶链式反应鉴定和表征“锌指”结构域。
DOI：
10.1073/pnas.88.2.671
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发表时间：
1991
影响因子：
11.1
通讯作者：
Berg,JM
中科院分区：
文献类型：
--
作者：
Pellegrino,GR;Berg,JM
我们发展了一种利用聚合酶链式反应扩增含有转录因子IIIA(Cys2-His2)型锌指序列串联阵列的DNA片段的方法。因为这些序列是以串联阵列的形式出现的，所以当使用从锌指结构域的氨基和羧基末端序列衍生的引物扩增时，就会产生条带的梯形。这个阶梯的“梯级”对应于编码一个锌指结构域、两个相邻的锌指结构域等的DNA片段。这是通过分离对应于n个锌指结构域的单独条带，并用相同的引物重新扩增它们来证明的。这产生了原始大小的带以及对应于1到(n-1)个锌指结构域的带。通过对一系列扩增产物进行克隆和测序，获得了这些条带编码锌指结构域的直接证据。由于所获得的序列缺乏冗余性，我们得出结论，每一条带对应于大量唯一的锌指编码序列。以来自不同来源的基因组DNA为模板的扩增反应混合物的结果进一步证明了锌指结构域在真核基因组中广泛而频繁地存在。我们认为，该方法是分离和鉴定锌指编码基因的一种有效技术。
We have developed a method for amplifying DNA fragments containing tandem arrays of "zinc-finger" sequences of the transcription factor IIIA (Cys2-His2) type by using the polymerase chain reaction. Because these sequences occur as tandem arrays, a ladder of bands is produced upon amplification using primers derived from the amino- and carboxyl-terminal sequences of a zinc-finger domain. The "rungs" of this ladder correspond to DNA fragments encoding one zinc-finger domain, two adjacent zinc-finger domains, and so on. This is demonstrated by isolating individual bands corresponding to n zinc-finger domains and reamplifying them with the same primers. This yields a band of the original size as well as bands corresponding to 1 through (n - 1) zinc-finger domains. Direct evidence that these bands encode zinc-finger domains was obtained by cloning and sequencing a collection of the amplification products. Due to the lack of redundancy in the sequences obtained, we conclude that each band corresponds to a large number of unique zinc-finger-encoding sequences. The results from amplification reaction mixtures using genomic DNA from a variety of sources as template provide further evidence that zinc-finger domains occur widely and frequently in eukaryotic genomes. We believe that this method is a powerful technique for the isolation and characterization of zinc-finger-encoding genes.