Applications of PCR in Natural Resources Research Lecture 2: The Process

1. TGERC OSU Applications of PCR in Natural Resources ResearchLecture 2: The Process Amy Brunner and Steve DiFazio FS599 Section 2 Oregon State University Department of Forest Science

2. 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

3. 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

4. The PCR Process: How it Works • 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: (click to activate) http://vector.cshl.org/resources/BiologyAnimationLibrary.htm

5. Molecular Markers:RandomAmplifiedPolymorphic DNA, AP-PCR • Structure Target Sequence = arbitrary primer (e.g. ggcattactc) • High Variability: Probably due to mutations in priming sequences • PCR-based method Amplify regions between priming sites by polymerase chain reaction Analyze PCR products by agarose gel electrophoresis. Marker is dominant (presence/absence of band). No prior sequence knowledge required Many variations on the theme (e.g., RAMP, ISSR)

6. Amplified Fragment Length Polymorphism • 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

7. 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)

8. Microsatellites (Simple Sequence Repeats) • Structure = Repeat (e.g., ga) Unique flanking regions • Number of repeats is highly variable among individuals Design primers ( ) complementary to flanking regions Amplify repeat region by polymerase chain reaction Analyze PCR products by polyacrylamide gel electrophoresis Marker is codominant and highly genetically informative

9. Fluorescent Labeling of Microsatellites Enables Multiplexing • 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

10. Single-Strand Conformational Polymorphism 1. Amplify Target Sequence • Highly sensitive to DNA sequence: can detect single base changes • Simple process but can be difficult to repeat 2. Denature product with heat and formamide 3. Analyze on native (nondenaturing) polyacrylamide gel 4. Base sequence determines 3-dimensional conformation, and rate of migration

11. Denaturing Gradient Gel Electrophoresis 1. Amplify Target Sequence 4. Denaturing gradient gels are difficult to produce: use perpendicular gradient to identify optimal conditions, move to CDGE: constant denaturant gel electrophoresis 2. Run product on gel with denaturing gradient (parallel or perpendicular to direction gel runs) 3. Product begins denaturing at a certain point, depending on base sequence: greatly retards migration and allows discrimination of alleles based on small sequence differences

12. Cleaved Amplified Polymorphic Sequence 1. Amplify Target Sequence • Fairly simple analysis (cutting can be a hassle) • Requires sequence information from several alleles (or luck) 2. Cut with a restriction enzyme that differentiates alleles X Allele 1 Allele 2 3. Alleles can be differentiated by size based on loss or gain of restriction site; May be able to analyze on agarose gel

13. 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

14. Allele Discrimination

15. Allele Discrimination Assay Overview

16. Example of a Quantitative PCR Application • Studying disease (Swiss Needle Cast) in Douglas Fir -L. Winton • Several plots of D. Fir express different levels of Swiss Needle Cast • Procedure to quantify amount of fungus • Extract DNA from D. fir needles • Design primers & fluorescent probe for single copy D. Fir gene • Design primers & fluorescent probe for single copy fungal gene • Perform Real-time PCR on DNA samples • Quantitative analysis: amount fungal DNA/ amount D. fir DNA

17. Choosing a Method • Molecular methods necessary? Which one? • Research question • Scale, both time and space (physiological, ecological, or evolutionary) • Choose the proper mutation rate • Level of discrimination (polymorphism) required • Materials at hand • Time and Money • Sequence information • Work in related species • Lab facilities

18. Comparing the Methods * Depends on cost of restriction enzymes employed

19. Some Research Problems and Appropriate Methods • Measure population structure and infer historic gene flow among populations (e.g., GST) • Need moderate polymorphism, low cost per sample • Allozymes, RAPD, CAPS (organellar), Microsatellites, AFLP, RFLP • Measure current gene flow by direct methods (e.g., maternity analysis, paternity analysis • Need high polymorphism, codominance, repeatability, low cost per sample, • Microsatellites, allozymes • Study disease resistance alleles in natural populations • Need rapid, low-cost, repeatable way to distinguish alleles • CAPS, SSCP, DGGE, TaqMan

20. Introduction of Our Research Problem • Black cottonwood (Populus trichocarpa) is a native riparian tree • Dioecious (separate sexes), prolific seed production, also extensive vegetative propagation • Large variation in flowering and leaf phenology • Observed large contiguous stands on islands with one sex predominating, synchronous flowering • Question: are these stands clonal? How extensive are clones in these populations?

21. Clatskanie 30 m Male Tree Female Tree