诺百文库服务.ppt

Novobio Custom ServicescDNA文库服务 Feb 2012 Chaozheng.xie@ 1 1. 文库概述 cDNA Library定义： A cDNA library refers to the complete, or nearly complete, set of all mRNAs contained with a cell or organism A cDNA library will contain a stable representation of the mRNA present in a specific tissue at the time of collection cDNA Library服务的目的： To isolate or preserve a gene of interest, for example a novel or disease gene cDNA libraries allow the study of tissue specific gene expression cDNA sequences gives the genetic relationship between organisms through the similarity of their DNA 注： cDNA文库仅包含正在表达的基因 不同物种、不同组织的cDNA文库不相同 ◆从基因库中筛选、分离基因，可据对待选基因相关信息的了解程度，确定筛选方法和条件。 ◆常用方法是利用一段核苷酸序列(DNA，cDNA或寡核苷酸)作探针(probe)，用放射性同位素或非放射性同位素标记探针，也可用抗体作探针，筛选基因库。 文库应用 - 筛选、分离基因 平板杂交筛选 克隆DNA转移到尼龙膜上；膜上的DNA在碱性条件下变性，解聚形成单链；再通过烤膜或紫外线照射，单链将与膜紧密结合；再将膜置于含有放射性标记的核酸探针的溶液中保温，使探针与其互补序列进行杂交；杂交后充分洗去未杂交的探针，然后可由放射性自显影鉴定。 寡聚核苷酸探针 抗体探针 表达筛选 通过抗体筛选来检测cDNA编码的蛋白质 cDNA文库构建的简明流程 以组织细胞中的mRNA为模板，反转录合成双链cDNA ，各cDNA分子分别插入载体形成重组子，再导入宿主细胞克隆扩增。 mRNA isolation, purification Check the RNA integrity Fractionate and enrich mRNA Synthesis of cDNA Treatment of cDNA ends Ligation to vector 2. 文库要点 Every good cDNA library has three key characteristics that distinguish it from mediocre counterparts: It is large enough to contain representatives of all sequences of interest, some of which may be derived from low-abundance mRNAs. QC: Total cfu; % recombinants 2. It includes a minimal number of clones that contain small (often defined arbitrarily as ≤500 bp) cDNA inserts. QC: AIS – Average Insert Size 3. It is composed of cDNA inserts that are near full-length copies of the mRNAs from which they were derived. QC: % Full Length (序列分析测序结果，看是否有完整ORF) 9 N 所需克隆数 P 要求的概率（0.99） 1/n 某一稀有mRNA在总mRNA中比例 如人约3万基因，约10万mRNA，要求某一单拷贝基因在文库中出现的概率为0.99时，文库的库容量最小是多少？ 文库QC - cfu 文库QC – Total cfu 倍比稀释, 涂LB平板，LB平板计数克隆形成数 文库QC – % recombinants AIS 随机挑取文库中的克隆，抽提质粒，进行PCR，以鉴定插入片段大小 文库QC – % Full Length 随机挑取文库中的克隆，进行测序 根据测序结果，进行ORF的预测，并结合核苷酸和预测蛋白序列有哪些信誉好的足球投注网站N