本科生《P》5-第五章目的基因制备与克隆策略3-10.pptVIP

Chapter 6 估计基因组文库大小的经验（empirical formula）公式： N=ln(1-P)/ln(1-a/b) 选择哪种载体用于基因组文库构建？ Which vector is used to choose for construction of genetic library? 建立基因组文库的一般程序： 1．载体DNA片段的制备 载体DNA分离纯化 限制酶切 脱磷酸化反应  Homopolymer tailing: the enzyme terminal transferase is used to add homopolymers of dA, dT, dG or dC to a DNA molecule. Pst I vector CCCCCCCC-3’ 3’-CCCCCCCC Insert CTGCAGGGGGGGG-3’ G 3’-GGGGGGGGACGTC G Pst I site GGGGGGGGACGTC CTGCAGGGGGGGG GACGT TGCAG CCCCCCCC CCCCCCCC Pst I site Enzyme digestion Terminal transferase + dC Pst I digestion Terminal transferase + dG DNA ligase Target gene ★Main advantage: 1) packaging in vitro may generate the recombinant phage, which increases the efficiency of the cloning process. 2) much easier to store and handle large numbers of phage clones than plasmids. 3 Cloning cDNA in bacteriophage vectors If a large number of recombinants was required, and a low-abundance mRNA was to be cloned, phage vectors may be more suitable. Cloning cDNA in λvectors using linkers: EcoR I methylase CCGAATTCGG GGCTTAAGCC Add EcoR I linkers ds cDNA Me Me CCGAATTCGG GGCTTAAGCC CCGAATTCGG GGCTTAAGCC CCGAATTCGG GGCTTAAGCC CCGAATTCGG GGCTTAAGCC CCGAATTCGG GGCTTAAGCC CCGAATTCGG GGCTTAAGCC Digest with EcoR I Ligate Insert vector vector LA λ vector RA ★genomic library: is often used to describe a set of clones representing the entire genome of an organism. To determine the introns To examine the control sequences responsible for regulating gene expression 二、 Cloning from genomic DNA 1 Genomic libraries ★aim of constructing a genomic library is for isolating a target DNA sequence. ★ Clone bank ( library ):A collection of independent clones is termed a clone bank or library N: the number of clones required P: desired probability of a particular sequence being represented(0.95 or 0.99) a: the average size of the DNA fragment to be cloned b: the size of the genome 9.3×102 2.1×103 1.4×104 Saccharomyces cerevisiae (yeast) 4.7×103 1.1×104 7