section G Gene manipulation.ppt

本文观看结束！！！ DNA ligation (DNA连接) Purpose: To insert a target DNA fragment into a vector. Function of DNA ligases: they will repair (ligate) a break in one strand of a dsDNA molecule. Energy sources: DNA ligase from E. coli uses NAD+ as energy source; T4 DNA ligase uses ATP (more commonly used). Different ends: Cohesive ends: Ligases are efficient at sealing the broken phosphodiester bonds for cohesive ends, Blunt ends: just T4 ligase can even ligate one blunt end to another, but with rather lower efficiency. Recombinant DNA molecules-I (重组DNA分子) Making of the recombinant DNA: Target DNA: ? may be a single fragment isolated from agarose gel, or ? a mixture of many fragments from genomic DNA. Vector DNA: the new vector DNA can be cut with the same enzyme. If the target DNA has been prepared by digestion with same enzyme (EcoRI). Recombinant molecules: The products are circular molecules with the target fragment inserted at the EcoRI site of the vector molecule with either orientation (方向). E E EcoRI E E S E E S 抗生素抗 性基因 Ori E A E B Recombinant DNA molecules-II (重组DNA分子) A problem: The recreation of the original vector plasmid, by circularization of the linear vector alone, is a competing side reaction which can make problems on the recombinant identification. Solution one--Paired distinct restriction: One solution is to prepare both the target DNA and the vector DNA using a pair of distinct restriction enzymes, such that they have non-compatible cohesive ends at either end. The likelihood (可能性) of ligating the vector into a circle is then much reduced. HO -AATTC HO -G G -OH CTTAA -OH Alkaline phosphatase (碱性磷酸酶) Solution two Linear vector: Alkaline phosph-atase removes phosphate groups from the 5-ends of DNA molecules. The linear vector will hence be unable to ligate into a circle, since no phosphates are available. Target DNA: A ligation with a target DNA insert can still proceed, since one phosphate is present to ligate one strand at each cut site. The