重叠重复人心钠素基因的化学合成及其克隆

Chemical Synthesis and Cloning of Concatenated Human Cardionation Genen

根据人心钠素的氨基酸序列,借助于计算机,设计了8个寡核苷酸片段,并在DNA自动合成仪上合成了这些片段。经5′磷酸化、退火、连接等操作,这些片段组装成由两个心钠素基因首尾以终止起始码TGATG相连的串联体。将它插入质粒pRC_(23)转化E.coli TAP_(106)后,获得pY X_(40)对重组子的酶切图谱、原位杂交及DNA序列的分析表明,插入基因的方向、读框正确。

Eight oligonucleotide fragments were designed with a computer and synthesized in amino acids sequence of human cardionatrin (ANF). Through annealing and ligation, these fragments were assembled into a concatenated gene consisting of two ANF genes ligated with TGATG, Then the concatenated gene was inserted into plasmid pRC23 and the reeombinant was tiansferred into E. coli TAP 106. Analysis of restriction enzyme mapping, hybridizing and DNA sequencing shows that the gene direction and reading frame are correct.