基因制备与克隆策略管理知识分析.pptVIP

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 估计基因组文库大小的经验（empirical formula）公式： N=ln(1-P)/ln(1-a/b) 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 organism Genome Size(kb) No.clones N,P=0.95 20kb inserts 45kb inserts Escherichia coli (bacteria) 4.0×103 6.0×103 2.7×103 Saccharomyces cerevisiae (yeast) 1.4×104 2.1×103 9.3×102 Arabidopsis thaliana (simple plant) 7.0×104 1.1×104 4.7×1