Xu B W, Paszty C, Lurquin P F
Washington State University.
Biotechniques. 1988 Sep;6(8):752-60.
A method is described which enables quantitative evaluation of the ability of gram-negative bacterial cells to perform homologous recombination between DNA molecules. This method is particularly useful in cases where the stringency of rec mutations is to be determined. The procedure is based on a wide-host-range vector (pRK404) in which two unequally truncated and overlapping fragments of the neo gene were cloned. When introduced into gram-negative bacteria either by transformation or by conjugation, molecules of this plasmid, pBX404-7, undergo unequal crossing-over leading to the restoration of a functional neo gene. The stringency of putative rec mutations can thus be determined by measuring the frequency at which kanamycin-resistant colonies appear in bacterial strains harboring pBX404-7.
本文描述了一种能够对革兰氏阴性细菌细胞在DNA分子间进行同源重组的能力进行定量评估的方法。该方法在确定rec突变的严格程度的情况下特别有用。该程序基于一个广宿主范围载体（pRK404），其中克隆了新霉素基因的两个不等长且重叠的片段。当通过转化或接合引入革兰氏阴性细菌时，该质粒pBX404 - 7的分子会发生不等交换，从而导致功能性新霉素基因的恢复。因此，通过测量携带pBX404 - 7的细菌菌株中出现卡那霉素抗性菌落的频率，可以确定假定的rec突变的严格程度。
