第二章l流am修改.ppt

GE HBGCHU LAM 核酸分离、纯化原则 保持核酸分子一级结构的完整性 分离核酸原则： 1）温度不要过高； 2）控制 pH 值范围( pH 值 5-9); 3）保持一定离子强度; 4）减少物理因素对核酸的机械剪切力. 5) 尽量简化操作步骤，缩短提取过程，以减少各种 有害因素对核酸的破坏 核酸的纯化应达到以下要求 1. 核酸样品中不存在对酶有抑制作用的有 机溶剂和过高浓度的金属离子； 2. 其它生物大分子如蛋白质、多糖和脂类 分子的污染应降到最低程度； 3. 排除其它核酸分子的污染，如提取DNA 分子时应去除RNA，反之亦然。 防止核酸的生物降解 细胞内或外来的各种核酸酶能消化核酸链中的磷酸二酯键，破坏核酸一级结构。 所用器械和一些试剂需高温灭菌，提取缓冲液中需加核酸酶抑制剂。 1 细胞的破碎 2 核蛋白的解聚、变性蛋白的去除 3 核酸的沉淀 4 核酸的浓度测定 5 核酸的保存 Applications Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of cloned DNA. Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer 四 Pulsed field gel electrophoresis Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid (DNA) molecules by applying an electric field that periodically changes direction to a gel matrix. At Columbia University in 1984, Schwartz and Cantor developed a variation on the standard protocol by introducing an alternating voltage gradient to improve the resolution of larger molecules. The procedure for this technique is relatively similar to performing a standard gel electrophoresis except that instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 60 degrees either side. The pulse times are equal for each direction resulting in a net forward migration of the DNA. Example of a real PFGE; drug resistant Staphylococcus aureus. The molecular weight markers are digested lambda phage (λ) and are given in kb SYBR Green I (SG) SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to DNA. The resulting DNA-dye-complex absorbs