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扩增噬菌体人基因组文库以形成多个亚文库并富集稀有目的克隆。利用人免疫球蛋白Cγ基因启动子、编码区设计的多对探针引物,PCR筛选已扩增的亚文库,获得共阳性亚文库;利用多条同位素标记的探针筛选上述共阳性亚文库,经相应探针引物PCR鉴定,获得共阳性多克隆噬菌斑;2000倍稀释共阳性多克隆噬菌斑稀释液,PCR筛选出阳性单克隆噬菌斑。基因转换到pBSK(+)载体测序,筛到长12 kb的人免疫球蛋白重链Cγ基因,含有启动子、编码外显子、穿膜外显子及polyA序列。与传统的单条探针筛库相比,新策略利用多条探针一次同位素标记,就筛到完整的稀有Cγ基因;利用PCR在亚文库、多克隆噬菌斑稀释液、单克隆噬菌斑稀释液三个层面上筛选,逐步缩减的筛选范围是一次同位素标记筛到完整稀有基因的前提。新策略不仅提高一倍工作效率,降低几倍的耗材,同时为需要长片段基因的功能基因组领域提供筛选捷径。
Phage human genomic libraries were amplified to create multiple sub-libraries and enriched for rare target clones. Using the human immunoglobulin Cγ gene promoter and multiple pairs of probe primers designed in the coding region, the amplified sub-library was screened by PCR to obtain a common positive sub-library; the multiple positive-sense sub-libraries were screened by using a plurality of isotope-labeled probes, Positive colonies were obtained by polyacrylamide gel electrophoresis (PCR) with the corresponding probe primers. The polyclonal positive colonies were screened by PCR with 2000-fold dilution. The gene was switched to the pBSK (+) vector to screen the human immunoglobulin heavy chain Cγ gene of 12 kb in length, which contained the promoter, coding exon, transmembrane exon and polyA sequence. Compared with traditional single-probe screens, the new strategy uses single-stranded labeling with multiple probes to screen out complete rare Cγ genes; PCR was performed in sub-libraries, polyclonal plaque dilutions, monoclonal plaques Dilution of the three levels of screening, and gradually narrowing the scope of screening is an isotope labeled prerequisite for complete rare gene. The new strategy will not only double your productivity, reduce your supplies several times, but also provide you with shortcuts for the functional genomics that require long fragment genes.