研究生课程：PCR技术的新进展及其应用(1).pptVIP

In this Lecture Principle of PCR PCR, the basics Optimize the PCR and troubleshoot Application of PCR Reverse-Transcription PCR（反转录PCR〕 PCR cloning（PCR克隆技术〕 Real-Time PCR（实时定量PCR〕 History of PCR Polymerase Chain Reaction PCR是一种体外酶促合成特定DNA片断的技术，是根据人类的需要对复杂生命过程的一种简单化的模拟。PCR技术的原理是DNA半保留复制。 DNA Replication in vivo The PCR Process in vitro The PCR in vitro PCR Optimization APPLICATIONS OF PCR PCR技术在基因克隆 基因表达差异分析中常用的方法 PCR cloning Semi-quantitative RT-PCR Real-Time PCR Cloning of PCR products Modified primers by adding RE sites at the 5’end of each primer T-A strategy: because some thermostatic DNA polymerase can add a non-template-depended A at the 3’end of PCR products, so can inserted the PCR products into a T-vector diractly Blunt-end PCR products cloning, many thermostatic DNA polymerase can amplify the DNA correctly, Inserted the DNA products into blunt-RE digested plasmid diractly. DNA聚合酶的特性及在基因克隆中的应用 Taq DNA聚合酶：某些耐热的DNA聚合酶，在DNA链延伸到模板DNA的末端时，并不是立即终止新链的延伸，而是在新链的3’末端加上一个A，形成突出的3’末端，为基因克隆提供了一个天然的粘末端。 PCR T-A cloning PCR cloning T-A strategy PCR 克隆：通过引物对克隆片断添加酶切位点 PCR 克隆：通过引物对克隆片断添加酶切位点 RT-PCR RTase: Avian myeloblastosis virus(AMV) and Moloney strain of murine leukemia virus(MMLV). Primers for first-stranded cDNA synthesis: Gene specific primers, Oligo(dT) primers for binding the poly(A) mRNA and Random hexamer primers RT-PCR To amplify cDNA copies of RNA To retrieve and clone the 5’ and 3’ terminus of RNA To generate cDNA library from a very small amounts of mRNA To identify the mutations and polymorphisms To measure the strength of gene expression (semi-quantitative RT-PCR) RT-PCR Full length cDNA synthesis with PCR RT-PCR RTase: Avian myeloblastosis virus(AMV) and Moloney strain of murine leukemia virus(MMLV). Primers for first-stranded cDNA synthesis: Gene specific primers, Oligo(dT) primers for binding the poly(A) mRNA and Random hexamer primers 基因表达差异的分析技术 Northern Blot Western blot Semi- quantatitive RT-PCR DNA Chip Protein Chip 二维电泳 质谱 Semi-quanti