本科生第五章目的基因制备与克隆策略详解演示文稿.pptVIP

Adaptors: are single-stranded non-complementary oligomers that may be used in conjunction with linkers, when annealed together and be added to the cDNA to provide sticky-end cloning without digestion of the linkers. OH-GATCCCCGGG DNA ligase CCCGGG GGGCCCCTAG-OH- 5’ GGGCCC GGATCC CCTAGG BamH I + OH-GATCCCCGGG GGGCCC 5’-OH-GATCCCCGGG GGGCCC CCCGGGGGGCCCHpaII 当前第31页\共有55页\编于星期三\6点 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 当前第32页\共有55页\编于星期三\6点 ★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. 当前第33页\共有55页\编于星期三\6点 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 当前第34页\共有55页\编于星期三\6点 ★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