TOTAL SYNTHESIS OF A GENE
TOTAL SYNTHESIS OF A GENE
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DOI：
10.1126/science.366749
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发表时间：
1979-01-01
期刊：
影响因子：
56.9
通讯作者：
KHORANA, HG
中科院分区：
文献类型：
--
作者：
KHORANA, HG
开发了用于全合成含有生物特异性序列的给定DNA的方法。双链形式的DNA被小心地分成短的单链片段，在互补链中具有适当的重叠。所有片段都是从受保护的核苷和单核苷酸开始化学合成的。然后使用[γ 32 P]ATP和多核苷酸激酶磷酸化适当寡核苷酸的5“-OH末端。然后允许几个到几个相邻的寡核苷酸在水溶液中形成双螺旋复合物，并且后者通过多核苷酸连接酶首尾相连以形成共价连接的双链体。随后短双链体的首尾连接产生总DNA。描述了用于构建生物学功能性抑制tRNA基因的方法。整个工作包括合成一个126个核苷酸长的双螺旋DNA，它对应于酪氨酸抑制tRNA的一个已知前体，启动子区和邻近C-C-A [C=胞嘧啶，A=腺嘌呤]末端的远端区的测序，它含有RNA转录物加工的信号，207个碱基对长的DNA的全合成，它包括控制元件，以及在2个末端的Eco R1限制性内切酶特异性序列，并通过合成基因的体外转录和体内琥珀抑制活性进行全面表征。
The method for the total synthesis of a given DNA containing biologically specific sequences was developed. The DNA in the double-stranded form is carefully divided into short single-stranded segments with suitable overlaps in the complementary strands. All the segments are chemically synthesized starting with protected nucleosides and mononucleotides. The 5''-OH ends of the appropriate oligonucleotides are then phosphorylated with the use of [.gamma.32P]ATP and polynucleotide kinase. A few to several neighboring oligonucleotides are then allowed to form bihelical complexes in aqueous solution, and the latter are joined end to end by polynucleotide ligase to form covalently linked duplexes. Subsequent head-to-tail joining of the short duplexes leads to the total DNA. The methods are described for the construction of a biologically functional suppressor tRNA gene. The total work involved the synthesis of a 126-nucleotide-long bihelical DNA correspnding to a known precursor to the tyrosine suppressor tRNA, the sequencing of the promoter region and the distal region adjoining the C-C-A [C=cytosine, A=adenine] end, which contained a signal for the processing of the RNA transcript, total synthesis of the 207 base-pair-long DNA, which included the control elements, as well as the Eco R1 restriction endonuclease specific sequences at the 2 ends and full characterization by transcription in vitro and amber suppressor activity in vivo of the synthetic gene.