Use of the DNA polymerase chain reaction for homology probing: isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species.
Use of the DNA polymerase chain reaction for homology probing: isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species.
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使用 DNA 聚合酶链式反应进行同源性探测：从多个物种中分离编码琥珀酸脱氢酶铁硫蛋白的部分 cDNA 或基因组克隆。
DOI：
10.1073/pnas.86.6.1934
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发表时间：
1989
影响因子：
11.1
通讯作者：
Scheffler,IE
中科院分区：
文献类型：
--
作者：
Gould,SJ;Subramani,S;Scheffler,IE
DNA聚合酶链反应是为了体外扩增特定的DNA序列而开发的，并且它已经在几个领域中用于各种各样的目的。我们已经开发了聚合酶链反应的应用，这是有用的部分cDNA或保守基因的基因组克隆的分离。我们使用这种技术克隆了编码琥珀酸脱氢酶（EC 1.3.5.1）的铁蛋白亚基（27 kDa）的基因，这些基因来自几个物种，包括人、大鼠、黑腹果蝇、拟南芥、粟酒裂殖酵母和酿酒酵母。在反应中，将对应于蛋白质的两个保守区域的混合寡核苷酸引物与基因组和cDNA模板结合使用。引物含有可编码相应肽序列的所有可能的核苷酸组合。这些寡核苷酸混合物分别含有262，144和8192个独特的序列.使用聚合酶链反应进行同源性探测允许人们利用比过滤器杂交筛选技术更复杂的寡核苷酸混合物作为探针。此外，聚合酶链式反应提供了直接合成DNA产物的优点，在某些情况下避免了构建cDNA或基因组文库的需要。聚合酶链反应的这种应用不仅可用于鉴定各种物种中的保守基因，而且可用于分离以前未知的基因家族成员。
The DNA polymerase chain reaction was developed for in vitro amplification of specific DNA sequences, and it has been used for a wide variety of purposes in several fields. We have developed an application of the polymerase chain reaction that is useful for the isolation of partial cDNA or genomic clones of conserved genes. We used this technique to clone the gene encoding the iron protein subunit (27 kDa) of succinate dehydrogenase (EC 1.3.5.1) from several species, including human, rat, Drosophila melanogaster, Arabidopsis thaliana, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Mixed oligonucleotide primers corresponding to two conserved regions of the protein were used in conjunction with genomic and cDNA templates in the reaction. The primers contained all possible nucleotide combinations that could encode the corresponding peptide sequences. These oligonucleotide mixtures contained 262,144 (2(18] and 8192 (2(13] unique sequences, respectively. Use of the polymerase chain reaction for homology probing allows one to utilize more complex mixtures of oligonucleotides as probes than is possible with filter hybridization screening techniques. In addition, the polymerase chain reaction offers the advantage of synthesizing the DNA product directly, in some cases obviating the need to construct cDNA or genomic libraries. This application of the polymerase chain reaction should be useful not only for the identification of conserved genes in a variety of species but also for the isolation of previously unknown members of gene families.