Applications of PCR in Natural Resources Research Lecture 2.ppt

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2016-08-25 发布于河南
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Applications of PCR in Natural Resources Research Lecture 2.ppt

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Applications of PCR in Natural Resources Research Lecture 2

Applications of PCR in Natural Resources ResearchLecture 2: The Process Schedule Lab on Saturday, November 20, 8:30-4:00 3083 Richardson Hall (south end near greenhouses) Demonstration of Genotyping Instrumentation Monday, Nov. 22, 4-4:20, Central Services Lab, ALS 3012 Results and Wrap-up Monday, Nov. 29, 5-5:30, 104 Peavy Outline Smorgasbord of PCR-based methods for detecting variation RAPD, AFLP, SSR, SNP, SSCP, DGGE, CAPS, TaqMan Choosing the appropriate method Some applications, including our research problem for the lab The PCR Process: How it Works Amplification of Specific Loci (Sequence Tagged Sites) The Advantages: You know what you’re amplifying, and often the source of the polymorphism Often more reliable than ‘anonymous’ methods: primer design can be very specific Targeting of specific genes (Expressed Sequence Tags) and genomes (i.e., chloroplast, nuclear, mitochondrial) Disadvantage: Labor-intensive to discover sequences Often rely on relatively few loci: vulnerable to violations of assumptions (e.g.,linkage equilibrium, mendelian inheritance, neutrality) Single-Strand Conformational Polymorphism Highly sensitive to DNA sequence: can detect single base changes Simple process but can be difficult to repeat Denaturing Gradient Gel Electrophoresis 4. Denaturing gradient gels are difficult to produce: use perpendicular gradient to identify optimal conditions, move to CDGE: constant denaturant gel electrophoresis Cleaved Amplified Polymorphic Sequence Fairly simple analysis (cutting can be a hassle) Requires sequence information from several alleles (or luck) Spectrofluorometric Thermal Cycler for Allele Discrimination Quantitative PCR CSL has an ABI PRISM 7700 Sequence Detection System Real-time detection: Each cycle produces a fluorescent signal proportional to the amount of PCR product present Allele Discrimination Allele Discrimination Assay Overview Example of a Quantitative PCR Application Studying disease (Swiss Needle Cast) in Douglas Fir