Recombineering: Chromosome Engineering by Homologous Rec

最近，我们已经开发了一个体内克隆和基因修饰系统，使用拟南芥介导的同源重组（红色）。该系统消除了用限制酶切割DNA或用DNA连接酶连接DNA片段的需要。我们相信，这种极其有效的重组系统将彻底改变重组DNA的工作方式。ssDNA寡聚物（短至20个碱基）和dsDNA寡聚物（短至30 bp）均可用于在细菌染色体、质粒或基因组BAC文库克隆上产生重组体。ssDNA寡核苷酸已用于在染色体上和BAC克隆中产生点突变、修复突变、产生缺失以及产生小插入。ssDNA的扩增仅需要λ噬菌体β蛋白，其结合电穿孔到细胞中的ssDNA寡核苷酸并使该核苷酸退火至其在细胞中的靶同源性。退火发生在复制过程中形成的ssDNA中间体上。长dsDNA（>1000bp）的扩增除了β之外还需要λ噬菌体功能Exo和Gam。Exo降解dsDNA的5'末端，产生β结合的ssDNA突出端。Gam抑制破坏进入的线性DNA的宿主核酸酶。

Recently, we have developed an in vivo cloning and gene modification system using lambda-mediated homologous recombination (Red). This system eliminates the need to cut DNA with restriction enzymes or join DNA fragments with DNA ligase. This extremely efficient recombination system will we believe, revolutionize the way in which recombinant DNA work is done. Both ssDNA oligos (as short as 20 bases) and dsDNA oligos (as short as 30 bp) can be used to generate recombinants on the bacterial chromosome, on plasmids, or on genomic BAC library clones. ssDNA oligos have been used to create point mutations, to repair mutations, to create deletions, and to create small insertions on the chromosome and in BAC clones. Recombination of ssDNA requires only the lambda phage Beta protein which binds the ssDNA oligonucleotide that is electroporated into cells and anneals that nucleotide to its target homology in the cell. Annealing occurs to the ssDNA intermediates that form during replication. Recombination of long dsDNA (>1000bp) requires lambda phage functions Exo and Gam in addition to Beta. Exo degrades 5' ends of dsDNA generating ssDNA overhangs for Beta to bind. Gam inhibits host nucleases which destroy the entering linear DNA.