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[单链合成DNA的克隆]

[Cloning of single-stranded synthetic DNA].

作者信息

Amirkhanov N V, Kuznedelov K D, Rivkin M I, Kumarev V P

出版信息

Bioorg Khim. 1985 Sep;11(9):1283-5.

PMID:2998406
Abstract

The method for cloning a single-stranded synthetic DNA with the short complementary oligonucleotides, that form corresponding restriction sites, is proposed. The potency of the method is demonstrated by cloning a single-stranded polynucleotide A (93 nucleotide residues (n. r.] in plasmid vector pBR327. The polynucleotide A includes a leader structure of the human fibroblast interferon gene. Oligonucleotides (IV) (20 n. r.) and (VI) (16 n. r.) were taken as strengthening complements and to create the sticky ends for the restrictases HindIII and EcoRI. 72% of the obtained clones appeared to be hybrid. Four hybrid clones were analyzed, and three of them carried the desirable insertion. The primary structures of these insertions are confirmed by sequencing.

摘要

提出了一种用短互补寡核苷酸克隆单链合成DNA的方法,这些寡核苷酸形成相应的限制性位点。通过在质粒载体pBR327中克隆单链多核苷酸A(93个核苷酸残基)证明了该方法的有效性。多核苷酸A包含人成纤维细胞干扰素基因的前导结构。寡核苷酸(IV)(20个核苷酸残基)和(VI)(16个核苷酸残基)用作增强互补物,并为限制性内切酶HindIII和EcoRI产生粘性末端。所获得的克隆中有72%似乎是杂种。分析了四个杂种克隆,其中三个携带了所需的插入片段。这些插入片段的一级结构通过测序得到了证实。

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