基因工程第五章（The fifth chapter of gene engineering）.docVIP

基因工程第五章（The fifth chapter of gene engineering） In vitro recombination and gene conversion in Chapter fifth The first section is connected DNA segments in vitro Section second of the recombinant was introduced into bacterial cells Connected to a sticky end, (Cohesive end ligation sticky ends connected): With sticky ends of two double stranded DNA molecules in DNA ligase, connected into a hybrid double stranded DNA. Advantage Time saving, convenient operation?; Easy recovery of exogenous fragment?; The problem Carriers ring? The insert can be inserted into the two? 2. different restriction enzyme connection at the end of the stick ? molecular carrier and gene has two identical restriction sites The vector and the target gene? Only one the same restriction enzyme recognition site No? The same restriction enzyme recognition sites, have isocaudarner The Sal fragment (CAGCTG) Xho fragment (GAGCTC) If you are not above?? At the end of two, flat connection Flat end joining (blunt end ligation): In the T4DNA ligase, the two has a flat end of the double stranded DNA molecules connected into DNA hybrid molecules. Advantage You can use any T4 ligase DNA flat end 5? - prominent sticky ends up The available Klenow polymerase to complete 3? - prominent sticky ends cut Available T4 DNA polymerase Xiaoping The main problem The connection efficiency is low ? high concentrations of substrate and T4 DNA ligase Adding coagulant: such as? It is not easy to recover the exogenous DNA fragment Two-way insertion Three, the modified sticky end connections Homopolymeric tail method?: Homopolymeric tail (homopolymer tails joining) is connected by terminal transferase in carrier and exogenous double stranded DNA 3ends with a oligonucleotid, made of artificial viscosity and then in the end, DNA ligase, connected into recombinant DNA. Advantage The effect of self loop does not occur; The higher connection efficiency; The problems Complex operation? ? gene difficult recovery May affect gene express