Intro to PCR The Polymerase Chain Reaction Photo courtesy of Fisher Scientific

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 Technique: For some applications, PCR and gel electrophoresis are the only steps required to perform analysis. For example, species in an unknown sample can be distinguished by banding patterns observed on a gel if the target amplified in PCR was a substantially different length for each species (typically 50 bp difference or greater). Diagram by Andy Vierstraete 1999

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 Technique: (1) Multiplex PCR uses more than one set of primers to amplify multiple targets in the same reaction. (2) Quantitative or Real-Time PCR uses fluorescent markers to track the number of copies of target sequence produced in each cycle. With careful calibration, the quantity of DNA present in the original sample can be extrapolated from the number of copies yielded.

Non-examples PCR is NOT used to: Amplify RNA or proteins Construct genomic or cDNA libraries Make monoclonal antibodies Conduct stem cell research Technique: If RNA is first reverse transcribed to create cDNA, PCR can be performed on the cDNA.

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

Thermal Cycler Steps Denature double-stranded DNA Anneal primers to single-stranded DNA Extend primers, yielding new double-stranded DNA Technique: Settings on the thermal cycler can be adjusted for reaction volume and number of cycles, as well as time and temperature of each of the following steps: (1) initial denaturation; (2) cyclic denaturation; (3) cyclic annealing; (4) cyclic extension; (5) final extension; and (6) post-reaction incubation. The total reaction will generally take 1 ½ to 3 hours. Cycling: Repeat steps 1 through 3 (20 - 40 times)

PCR Animation--3D

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

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

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

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

Roles of PCR Reagents GoTaq® PCR Mix Taq polymerase 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 loading dye Adds color and viscosity for future gel loading

Roles of PCR Reagents Primers DNA template 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

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

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

Setting Up the Reaction PCR reagent volumes vary by experiment An example ITS amplification in 25 ul final volume is.. Reagent Reagent concentration Final concentration Volume/ reaction # of reactions = 10 GoTaq ® Green PCR mix 2X 1X 12.5 ul 125 ul ITS forward primer 10 M 0.3 uM 0.75 ul 7.5 ul ITS reverse primer 10 uM Water -- 1.0 ul 10 ul Total Master Mix 15.0 ul 150 ul Techinique: Most reactions vary in size from 10 ul to 50 ul. Larger reactions are considered to be more robust because a larger quantity of each reagent is present. (Note that larger reactions increase the cost.)

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 Technique: When attempting an amplification new to you, find a previous study of a similar organism or tissue and match conditions.

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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

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

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

California State Investigation and Experimentation Standards 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

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