Mazin A V, Dianov G L, Safronov P F, Salganic R I
Mol Biol (Mosk). 1984 Jul-Aug;18(4):1081-9.
A method is suggested for chemical modification of preselected regions of plasmid DNA by complementary single-stranded restriction fragments of DNA (srf DNA), carrying alkylating reagents. The gene coding for tetracycline resistance of plasmid pBR322 was used as a target. Srf DNA was prepared by a partial digestion of a double-stranded EcoRI-BamHI restriction fragment (377 base pairs) from Tcr by E. coli exonuclease III. The residues of an alkylating reagent N,N,N'-tri(beta-chlorethyl)-N'-(p-formylphenyl) propylenediamine 1,3 (TFP) were attached covalently to 4-5% of sfr DNA bases. The alkylating derivative of the sfr DNA was hybridized with supercoiled pBR322 plasmid DNA. The hybridization conditions (37 degrees C, 40% formamide, 0,2 M NaCl, 0,1 M Tris-HCl pH 7,5, 0,001 M EDTA) under which the bases carrying TFP residues are not eliminated from the sfr DNA, and transforming activity of pBR322 DNA does not decrease were established. It was shown that about 20% of plasmid pBR322 molecules form D-loops with alkylating sfr DNA under these conditions. It was shown that sfr DNA, carrying TFP can alkylate the complementary region of plasmid DNA, forming cross-linked D-loops. A method for the site-directed mutagenesis of switching off the preselected genes or non-transcribed DNA functional regions (promotors, introns etc) integrated into plasmids of other vectors is suggested.
本文提出了一种通过携带烷基化试剂的DNA互补单链限制性片段（srf DNA）对质粒DNA预选区域进行化学修饰的方法。以质粒pBR322的四环素抗性编码基因作为靶标。通过用大肠杆菌核酸外切酶III对来自Tcr的双链EcoRI - BamHI限制性片段（377个碱基对）进行部分消化来制备srf DNA。烷基化试剂N，N，N'-三（β-氯乙基）-N'-（对甲酰苯基）丙二胺1,3（TFP）的残基共价连接到4 - 5%的sfr DNA碱基上。将sfr DNA的烷基化衍生物与超螺旋pBR322质粒DNA杂交。确定了杂交条件（37℃，40%甲酰胺，0.2M NaCl，0.1M Tris - HCl pH 7.5，0.001M EDTA），在此条件下携带TFP残基的碱基不会从sfr DNA中消除，并且pBR322 DNA的转化活性不会降低。结果表明，在这些条件下，约20%的质粒pBR322分子与携带烷基化试剂的sfr DNA形成D环。结果表明，携带TFP的sfr DNA可以使质粒DNA的互补区域烷基化，形成交联的D环。本文还提出了一种定点诱变方法，用于关闭整合到其他载体质粒中的预选基因或非转录DNA功能区域（启动子、内含子等）。
