Gordhan B G, Parish T
Methods Mol Med. 2001;54:77-92. doi: 10.1385/1-59259-147-7:077.
Gene replacement by homologous recombination (HR) is an invaluable tool in understanding the physiology and the significance of specific genes in the virulence of Mycobacterium tuberculosis. It will also allow for the development of rationally attenuated strains as candidate vaccines to prevent the spread of tuberculosis. Classically, allelic replacement involves the introduction of nonreplicating DNA (suicide plasmids) carrying a mutated copy of the targeted gene, most often disrupted by an antibiotic resistance determinant, into the chromosome. A single recombination event (cross-over) between the two alleles will result in integration of the entire plasmid to generate a single crossover (SCO) strain carrying both wild-type and mutated copies of the gene. If two recombination events occur, a double cross-over (DCO) is generated where the wild-type allele is replaced by the mutant allele. Strains with an SCO can also give rise to DCO strains when a second recombination event takes place (Fig. 1).
通过同源重组（HR）进行基因替换是了解结核分枝杆菌毒力中特定基因的生理学和意义的一项宝贵工具。它还将有助于开发经过合理减毒的菌株作为候选疫苗，以预防结核病的传播。传统上，等位基因替换涉及将携带靶向基因突变拷贝的非复制性DNA（自杀质粒）导入染色体，该突变拷贝通常被抗生素抗性决定簇破坏。两个等位基因之间的单个重组事件（交叉）将导致整个质粒整合，从而产生携带该基因野生型和突变型拷贝的单交换（SCO）菌株。如果发生两个重组事件，则会产生双交换（DCO），其中野生型等位基因被突变等位基因取代。当发生第二次重组事件时，具有SCO的菌株也可以产生DCO菌株（图1）。
