【大学】常用分子生物学技术的原理及应用教程.pptVIP

常用分子生物学技术的原理及应用 The popular technology in molecular biology: principle and application 第 一 节分子杂交与印迹技术Molecular hybridization  blotting technology （一）Southern blotting Northern blotting　结果 蛋白质的转移只有靠电转移。 蛋白质的检测以抗体作探针。 用碱性磷酸酶（ALP）、辣根过氧化酶（HRP）标记第二抗体，底物显色来检测蛋白区带的信号，底物亦可与化学发光剂结合以提高敏感度。 或用同位素标记第二抗体，放射自显影法检测蛋白区带的信号。 斑点印迹 (dot blotting) A Dot blot is a technique in molecular biology used to detect biomolecules. It represents a simplification of the northern blot, southern blot, or western blot methods. In a dot blot the biomolecules to be detected are not first separated by electrophoresis. Instead, a mixture containing the molecule to be detected is applied directly on a membrane as a dot. This is then followed by detection by either nucleotide probes (for a northern blot and southern blot) or antibodies (for a western blot). The technique offers significant savings in time, as chromatography or gel electrophoresis, and the complex blotting procedures for the gel are not required. However, it offers no information on the size of the target biomolecule. Furthermore, if two molecules of different sizes are detected, they will still appear as a single dot. Dot blots therefore can only confirm the presence or absence of a biomolecule or biomolecules which can be detected by the DNA probes or the antibody. 原位杂交 (in situ hybridization) In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a section of tissue, or, if the tissue is small enough, in the entire tissue. DNA ISH can be used to determine the structure of chromosomes. Fluorescent DNA ISH (FISH) can, for example, be used in medical diagnostics to assess chromosomal integrity. RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts. 第 二 节 PCR技术的原理与应用 三、几种重要的PCR衍生技术 （一）反转录PCR技术 (RT-PCR) （二）原位PCR技术 (ISP) （三）实时PCR技术