Mazin A V, Saparbaev M K, Ovchinnikova L P, Dianov G L, Salganik R I
Institute of Cytology and Genetics, Siberian Department of the USSR Academy of Sciences, Novosibirsk.
DNA Cell Biol. 1990 Jan-Feb;9(1):63-9. doi: 10.1089/dna.1990.9.63.
A new site-directed method for inserting long single-stranded DNA fragments into any region of a duplex vector is described. Its major advantage is independence of the location of the restriction sites. The method involves the assembly of single-stranded DNA fragments by ligation to both ends of the inserted fragments of two cohesive flanks that are complementary to the target region. Short oligonucleotide templates are used to direct the ligation. The resulting fragments, designated further as omega fragments with cohesive flanks, are hybridized with a gapped DNA vector. The heteroduplexes are transformed into Escherichia coli cells without enzymatic filling and sealing of gapped DNA. As a consequence of intracellular repair and heteroduplex resolution, insertion mutants are recovered. To demonstrate the method's efficiency, we inserted a 51-nucleotide synthetic DNA fragment containing a modified glucocorticoid receptor binding site into the region of pBR322, near the transcription starting point of the tet gene. The method we developed makes possible site-directed insertion of synthetic and genome-derived DNA fragments at least 200 nucleotides long.
本文描述了一种将长单链DNA片段插入双链载体任意区域的新的定点方法。其主要优点是不受限制酶切位点位置的影响。该方法包括通过连接与靶区域互补的两个粘性侧翼的插入片段的两端来组装单链DNA片段。短寡核苷酸模板用于指导连接。所得片段，进一步称为具有粘性侧翼的ω片段，与缺口DNA载体杂交。异源双链体在没有对缺口DNA进行酶促填充和封闭的情况下转化到大肠杆菌细胞中。由于细胞内修复和异源双链体分辨率，获得了插入突变体。为了证明该方法的效率，我们将一个含有修饰的糖皮质激素受体结合位点的51个核苷酸的合成DNA片段插入到pBR322的tet基因转录起始点附近的区域。我们开发的方法使得至少200个核苷酸长的合成和基因组来源的DNA片段的定点插入成为可能。
