分子生物学英文课件：molecular biotechnology.ppt

6. Derivative PCR RT-PCR (reverse transcription PCR) real time PCR (1) RT-PCR Combination of RNA reverse transcription and PCR reaction. Excellence: quantitative and qualitative analysis of RNA. (2) Real time PCR Quantitative PCR a pair of special primer (labeled fluorescence group, R and Q) as probes In a real time PCR assay, a positive reaction is detected by accumulation of a fluorescent signal. The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (ie exceeds background level). Ct levels are inversely proportional to the amount of target nucleic acid in the sample (ie the lower the Ct level the greater the amount of target nucleic acid in the sample). * 聚合酶链式反应。可将微量目的DNA片断扩增100万倍以上。敏感度高，特异性强，产率高，重复性好，快速，简便。K. mullis, 1993年诺贝尔奖。 * 体外扩增某一特异DNA片段的方法。原理：半保留复制。 * 最早，就在水浴锅里实现扩增。 * 与其他技术结合，发展出许多种PCR技术，进一步提高了PCR反应的特异性和应用的广泛性。 * Molecular Biotechnology Section I Molecular Hybridization and Blotting Technique A phenomenon or technique of forming a heteroduplex from two complementary polynucleotide strands from different sources (DNA and DNA; DNA and RNA; RNA and RNA) 1. Molecular Hybridization Related conceptions/principles DNA denaturation DNA renaturation 2. Probe DNA or RNA fragment labeled with radioisotope, biotin or fluorescent is used to detect specific nucleic acid sequences by hybridization. DNA probe RNA probe 3. Blotting Transfer (blot) biologic macromolecules separated in the gel and fix them to nitrocellulose/nylon membrane by diffusion, electro-transferring or vacuum absorption, then detect it. 1975, Edwen Southern Blotting is widely used for detecting the presence of specific macro- molecules (proteins, mRNAs or DNA sequences) in a mixture. (1) Southern blotting Genomic DNA (from tissues or cells) are cut by RE, separated by gel electro- phoresis and denatured in solution, then transferred to a nitrocellulose membrane for dete