基因克隆表达及功能研究1 课件.pptVIP

Gene cloning, expression and functional study 基因克隆，表达及功能研究;vectors; Cloning vectors;Cloning vectors: allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level. ;Contains an origin of replication, allowing for replication independent of host’s genome. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistance blue-white screening Contains a multiple cloning site (MCS) Easy to be isolated from the host cell.;Ampicillin resistant? yes yes Tetracycline resistant? No yes;;Blue white screening;Recreated vector: blue transformants Recombinant plasmid containing inserted DNA: white transformants;Multiple cloning sites;A plasmid vector for gene expression;T7 promoter;; Bacteriophage vector; viruses that can infect bacteria. 48.5 kb in length Linear or circular genome (cos ends) Lytic phase (Replicate and release) Lysogenic phase (integrate into host genome);Analysis of eukaryotic genes and the genome organization of eukaryotes requires vectors with a larger capacity for cloned DNA than plasmids or phage ?. Human genome (3 x 109 bp): large genome and large gene demand vectors with a large size capacity. ;Cosmid vectors;Digestion;YAC vectors;Essential components of YAC vectors : Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) for proliferation in the host cell. ampr for selective amplification and markers such as TRP1 and URA3 for identifying cells containing the YAC vector in yeast cells. Recognition sites of restriction enzymes (e.g., EcoRI and BamHI) ;YAC Cloning;BAC vectors 细菌人工染色体 1. More stable than YAC 2. Capacity is 300-350 kb 3. One to two copies in each cell 4. Easy to handle 5. More popular in genomic mapping;I1 Genomic libraries ;;I1-1 Representative gene libraries ;I1-2 Size of library (ensure enough clones);For example :for a probability of 0.99 with insert sizes of 20 kb these values for the E.coli (4.6×10