第八讲、第九讲-2006.ppt

Cloning strategies克隆策略 三、连接、包装和文库的扩增 ligation, packaging and amplification of libraries 酶切后通过密度梯度离心或电泳分离17~23Kb的片段用于连接. after digestion the sample is fractionated, either by density gradient centrifugation or by electrophoresis. Fragments in the range 17-23kb can then be selected for ligation. Sau3A、MboⅠ---同裂酶---基因组DNA。 BamHⅠ---同尾酶---载体 插入片段可用碱性磷酸酶处理，以减少自连或串联体的形成。 载体用BamHⅠ和SalⅠ处理除去填充片段。 Sau3A or MboⅠ(recognized the same sequence,) digested fragments can be inserted into the BamHⅠsite of a vector such as EMBL4. self-ligation or concatemer formation. stuffer fragment. phosphatase 可采用EcoR I 回收DNA片段。 连接时将产生由cos位点隔开的串联DNA分子，可作为包装底物。 The EcoR I site in the vector can be used to excise the insert after cloning. When ligation is carried out, concatemeric recombinant DNA molecule are produced, which are suitable substrates for packaging in vitro. 用异丙醇沉淀，来自填充片段的小DNA在上清夜中。 The short fragments can be removed from the preparation by precipitation with isopropanol, which leaves the small fragments in the supernatant. 接头由两条寡核苷酸单链构成，具有双链区和单链粘性末端。 接头与cDNA连接后可以直接与载体连接，不用封闭内部的酶切位点。 adaptors are single-stranded non-complementary oligomers that may be used in conjunction with linker. When annealed together, a linker/adaptor with one blunt end and one sticky end is produced, which can be added to the cDNA to provide sticky-end cloning without digestion of the linkers. A BamHⅠadaptor 5`-GATCCCCGGG-3` is annealed with a single-stranded HpaⅡ linker 3`-GGGCCC-5` to generate a double stranded sticky-ended molecule. ? 5`-GATCCCCGGG-3` 3`-GGGCCC-5` 5`-端为羟基，不带磷酸基团。 3，利用同聚尾进行连接，use homopolymer tailing to ligate 用末端转移酶加同聚尾。cDNA可通过dG-dC加尾克隆至质粒载体的PstⅠ位点。 the enzyme terminal transferase is used to add homopolymers of dA, dT, dG or dC to a DNA molecule. ? cDNAs can be cloned into the PstⅠsite of a plasmid vector by dG-dC tailing. 同聚尾连接有两个优点： G和C同聚尾可以形成很长的退火区，能够形成带有四个缺口的环状DNA分子，导入大肠杆菌细胞后可以在细胞内