宏基因组文库构建.pptVIP

宏基因组文库构建 威斯腾生物技术中心 400-675-6758 什么是文库？ Library 基因文库（也称DNA文库）是指某一生物体全部或部分基因的集合，就像一个没有目录的“基因图书馆” 基因文库的组成： 外源DNA片段：研究对象基因组DNA或cDNA 载体：质粒载体（plasmid）、黏粒载体（cosmid）、人工染色体（BAC、YAC）等 宿主：大肠杆菌（E.coli）、酵母（Saccharomyces cerevisiae） 什么是宏基因组文库？ 从环境样品中直接提取总DNA，经纯化后部分酶切，然后把这些DNA片段连接到载体上，转化替代宿主细胞，形成一个重组DNA文库即宏基因组文库。获得的克隆子根据宿主细胞获得的新功能进行筛选, 或用相关已知序列设计探针或PCR引物寻找并分离目的基因片段，加上表达调控元件后使目的基因表达，从而获得产物。 什么是宏基因组文库？ Obtaining bacteria in pure culture is typically the first step in investigating bacterial processes. standard culturing techniques account for 1% or less of the bacterial diversity in most environmental samples a suite of culture-independent techniques are needed to complement efforts to culture the thousands or millions of unknown species in the environment. sequencing of ribosomal RNAs and the genes encoding them was introduced to describe uncultured bacteria in the environment. The most startling result of the many microbial diversity studies that have employed 16S RNA culture-independent methods is the richness of the uncultured microbial world. 什么是宏基因组文库？ Describing the phylogenetic diversity of uncultured microorganisms is only the first step. assign ecological roles to them. Exploiting the rich microbial biodiversity for enzyme and natural product discovery is an active research area that has been reviewed elsewhere. Metagenomics Defined Metagenomics describes the functional and sequence-based analysis of the collective microbial genomes contained in an environmental sample. Many environments have been the focus of metagenomics, including soil, the oral cavity, feces, and aquatic habitats, as well as the hospital metagenomics. Extract DNA from soil sample A 每份土样加入lysis buffer（750μl/g），悬浮震荡均匀； B 加入溶菌酶（200μl/g），混匀； C 37℃温育2h，每半小时颠倒混匀一次； D 加入20%SDS（200μl/g）， 70℃水浴2h，每半小时颠倒混匀一次； E 5000rpm，30min 离心，保留上清，重复收集上清1~2次； F 加入约1/3体积PEG/NaCl，4℃静置过夜； G 5000rpm，30min离心； 弃上清，75%酒精洗涤2~3次； H待干燥后，加入适量TE溶解； I 提取得到的DNA过PVPP柱（可以在3000 rpm离心过柱）， 提取得到的DNA可储存在-20℃。 脉冲电泳分