TOTAL SYNTHESIS OF A GENE FOR BOVINE RHODOPSIN
TOTAL SYNTHESIS OF A GENE FOR BOVINE RHODOPSIN
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DOI：
10.1073/pnas.83.3.599
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发表时间：
1986-02-01
影响因子：
11.1
通讯作者：
OPRIAN, DD
中科院分区：
文献类型：
--
作者：
FERRETTI, L;KARNIK, SS;OPRIAN, DD
为了对牛视紫红质进行系统的结构-功能研究，我们完成了其1057个碱基的基因全合成。合成基因含有28个独特的限制性位点，平均间隔60个碱基对。用含有所需核苷酸变化的合成对应物替换特异性限制性片段，可在基因的所有部分进行特异性诱变。该基因的合成涉及总共72个合成寡核苷酸（15-40个核苷酸长）的酶促连接以形成DNA双链体。总基因由克隆的三个合成片段组装而成。所有合成的寡核苷酸通过5“-末端分析表征，并且连接反应的准确性通过对三个片段以及完整基因进行测序来确认。
To carry out systematic structure-function studies of bovine rhodopsin by specific amino acid replacements, we have accomplished the total synthesis of its gene, which is 1057 base pairs long. The synthetic gene contains 28 unique restriction sites that are on the average 60 base pairs apart. Replacement of specific restriction fragments by synthetic counterparts containing the desired nucleotide changes permits specific mutagenesis in all parts of the gene. The synthesis of the gene involved enzymatic joining of a total of 72 synthetic oligonucleotides, 15-40 nucleotides long, to form DNA duplexes. The total gene was assembled from three synthetic fragments that were cloned. All synthetic oligonucleotides were characterized by 5''-end analysis, and the accuracy of the joining reactions was confirmed by sequencing the three fragments as well as the complete gene.