Intro to PCR The Polymerase Chain Reaction

1. Intro to PCRThe Polymerase Chain Reaction Photo courtesy of Fisher Scientific

2. Definition Polymerase Chain Reaction (PCR): A procedure to amplify a specific DNA region • Yields millions of copies of the target region • Makes enough DNA for further molecular work • Is the first step in preparing DNA for: • Sequencing • Restriction digestion • Bacterial cloning Diagram by Andy Vierstraete 1999

3. Application Examples • PCR is commonly used to… • Identify species • Identify alleles/genotypes to assess variability in a population • Create sequences for phylogenies to determine taxonomic relationships • Conduct forensic investigations

4. Non-examples • PCR is NOT used to: • Amplify RNA or proteins • Construct genomic or cDNA libraries • Make monoclonal antibodies • Conduct stem cell research

5. Quick Quiz The purpose of PCR is to: • make copies of a specific region of DNA • identify the presence of particular genes • make sufficient genetic material for future molecular work • all of the above

6. Thermal Cycler Steps Denature double-stranded DNA Anneal primers to single-stranded DNA Extend primers, yielding new double-stranded DNA Cycling:Repeat steps 1 through 3 (20 - 40 times)

7. PCR Animation--3D

8. Quick Quiz A thermocycler protocol is comprised of which series of steps? • Decontamination, amplification, excision • Annealing, polymerization, cooling • Transcription, translation, expression • Denaturing, annealing, extension

9. Quick Quiz PCR amplifies molecular products in a(n) __________ fashion: • Logarithmic • Exponential • Linear • Random

10. Quick Quiz Primers: • Stabilize double stranded DNA • Are enzymes that catalyze the copying process • Are short single-stranded DNA fragments • Are long strings of nucleotides

11. Laboratory PCR Steps(Steps completed by researcher) Create Master Mix of reagents and aliquot into tubes Add DNA template(s) Program thermal cycler, load with tubes and start Remove tubes and analyze results

12. Roles of PCR Reagents • GoTaq® PCR Mix • Taqpolymerase • Enzyme that extends growing DNA strand complementary to DNA template • MgCl2 • Provides ions needed for enzyme reaction • dNTP’s • Nucleotides (Adenine, Cytosine, Guanine, Thymine) building blocks for new DNA strands • Buffer • Maintains optimal pH for enzyme • Green loadingdye • Adds color and viscosity for future gel loading

13. Roles of PCR Reagents • Primers • Anneal to single-stranded DNA template • Provide initiation site for extension of new DNA • Forward primer • Anneals to DNA anti-sense strand • Reverse primer • Anneals to DNA sense strand • DNA template • In this case, the product of our DNA extraction

14. Quick Quiz Which of the following reagents is NOT in a master mix? • MgCl2 • Template DNA • H2O • dNTPs

15. Quick Quiz If you forgot to add one of your primers your resultant gel will probably have • No bands • A smear • A band of the wrong size • Many bands

16. Setting Up the Reaction • PCR reagent volumes vary by experiment • An example ITS amplification in 25 ul final volume is..

17. Considerations • Contamination can easily lead to erroneous results • Avoid contaminating with DNA or PCR product… • DNA stocks, PCR reagents • Gloves, tips, pipetters, benches • Carefully measure reagent quantities • Use appropriate cycling conditions

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19. Resources • http://www.dnai.org/text/mediashowcase/index2.html?id=582 • Promega PCR Protocols & Reference • http://www.promega.com/guides/pcr_guide/ • Promega GoTaq Kit Handbook • http://www.promega.com/tbs/9pim712/_row/9pim712_row.pdf • Additional photos from Cornell University • http://www.igd.cornell.edu/MolecularMarkers/PCR%20basics.pdf

20. California State Chemistry Standards Grade 8 5a. Reactant atoms and molecules interact to form products with different chemical properties 6c. Living organisms have many different kinds of molecules… Grades 9-12 2.a. Atoms combine to form molecules by sharing electrons 2b. Chemical bonds between many large biological molecules are covalent 2c. Salt crystals are repeating patterns of positive and negative ions held together by electrostatic attraction 6. Solutions are homogenous mixtures of two or more substances 8. Chemical reaction rates depend on factors that influence the frequency of collision of reactant molecules

21. California State Biology Standards Grade 7 2e. DNA is the genetic material of living organisms and is located in the chromosomes of each cell 3a. Genetic variation and environmental factors are causes of evolution and diversity of organisms Grades 9-12 1b. Enzymes are proteins that catalyze biochemical reactions without altering the reaction equilibrium 1d. Molecular biology outlines the flow of information from transcription of RNA to translation of proteins 1h. Most macromolecules in cells and organisms are synthesized from a small collection of simple precursors 2. Mutation and sexual reproduction lead to genetic variation in a population 4. Genes are a set of instructions encoded in DNA… 5a. General structure and function of DNA, RNA and proteins 5b. Base-paring rules, copying of DNA, replication, transcription 5d. Basic DNA techonolgy

22. California State Investigation and ExperimentationStandards Grades 7 b. Select and use appropriate tools and technology to perform tests, collect data, analyze relationships, and display data Grade 8 a. Plan and conduct a scientific investigation to test a hypothesis Grades 9-12 a. Select and use appropriate tools and technology to perform tests, collect data, analyze relationships, and display data l. Analyze situations and solve problems that require combining and applying concepts from more than one area of science

23. National Standards Grades 6-12 Content Standard A: Science as Inquiry Content Standard C: Life Science Content Standard E: Science and Technology