cosmid克隆筛选的体内同源重组法——一个含有小鼠t复合体连锁DNA顺序的cosmid克隆的分离

The Selective Isolation of Cosmid Clones by Homologous Recombination in Escherichia coli ——A Cosmid Clone Containing t Complex Linkage DNA Sequence of Mouse Waa Isolated

一个从cosmid分子克隆库中筛选特别基因顺序的遗传学方法——体内同源重组(invlvo homologous recombination)法。即使探针DNA与分子克隆库中带有与探针同源顺序的克隆发生体内重组,然后以遗传学方法进行筛选。cosmid分子克隆库构建在rec宿主细胞内,经体内包装(in vivo Packaging)成λ噬菌体颗粒,把该噬菌体颗粒转入带有探针DNA的rec^+细胞内,探针是已被克隆在与cosmid载体没有同源顺序的质粒(如PUC8或PUC9)内的。经过一段时间(1—3小时),待重组发生后,把cosmid进行体内包装。此时探针DNA连同质粒已整合入cosmid基因组内,因此它带有原为两个载体所分别带有的双重抗性——Amp^r(氨苄青霉素,PUC8或PUC9)和Kan^r(卡那霉素,cosmid)。这种双重抗性菌落可在含有这2种抗菌素的培养平皿上选出,该重组cosmid借助于λ切除酶的作用将已被整合的探针质粒重新切除,再经体内包装后,该cosmid被还原并纯化,然后可用一含有Xgal的培皿识别和选出。本文用此法以有关DNA探针从cosmid分子克隆库中分离得到含有与小鼠t复合体连锁的基因组顺序的克隆,并对该克隆作了物理图谱分析。

A procedure for the selective isolation of specific cosmid clones by homologous recombination between cosmid clones of genomic library and a probe DNA sequence cloned in a plasmid in vivo has teen developed. The cosmid library was constructed in a rec- host cell strain and packaged into phage particles in vivo. The rec+ host cells containing a DNA sequence used as selection probe cloned in (he pUC plasmid were infected by packaged cosmid pliage particles. There is no homology between cosmid and the plasmid vectors. After a period of 1-3hr. for the recombination to take place, the probe plasmids were integrated into cosmid, in which the DNA sequence are homologous with the probe, by homologous recombination. The cosmids are then packaged in vivo and transferred imo a rec' cell strain. The specific cosmid clones were selected by double antibiotic resistance carried by both vectors. The probe plasmid can be excised by X excision enzyme by using superinfection with red+ phage. After packaging in vivo, these cosmid revertants can be identified on Xgal plate.A cosmid clone containing the t complex linkage DNA sequence of mouse was selected by using the procedure above with a prohe derived from microdissected metaphase chromosome.