Applications of PCR in Natural Resources Research Lecture 2课件.pptVIP

Applications of PCR in Natural Resources ResearchLecture 2: The Process;Schedule;Outline;Chain reaction relies on DNA replication process Repeated cycles of melting (strand separation), primer annealing, and primer extension by cycling temperatures Need a tough enzyme to deal with high temperatures Polymerases isolated from thermophilic bacteria (Thermus aquaticus, Pyrococcus furiosus) The Process: ;= arbitrary primer (e.g. ggcattactc);Polymorphism based on gain or loss of restriction site, or selective bases Technically demanding and expensive Many markers generated, mostly dominant More reliable than RAPD, less so than SSR No prior sequence knowledge required ;Amplification of Specific Loci (Sequence Tagged Sites);Amplify repeat region by polymerase chain reaction;Acrylamide gel with 5 microsatellite loci and internal size standard Four or more colors can be distinguished on many fluorimagers; coupled with size discrimination allows simultaneous analysis of nine or more loci;Single-Strand Conformational Polymorphism;Denaturing Gradient Gel Electrophoresis;Cleaved Amplified Polymorphic Sequence;Spectrofluorometric Thermal Cycler for Allele Discrimination Quantitative PCR;Allele Discrimination;Allele Discrimination Assay Overview;Example of a Quantitative PCR Application;Choosing a Method;Comparing the Methods;Some Research Problems and Appropriate Methods;Introduction of Our Research Problem;30 m