Presentation on theme: "Intro to PCR The Polymerase Chain Reaction"— Presentation transcript:
1 Intro to PCR The Polymerase Chain Reaction Photo courtesy of Fisher Scientific
2 Definition Polymerase Chain Reaction (PCR): A procedure to amplify a specific DNA regionYields millions of copies of the target regionMakes enough DNA for further molecular workIs the first step in preparing DNA for:SequencingRestriction digestionBacterial cloningTechnique: 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
3 Application Examples PCR is commonly used to… Identify species Identify alleles/genotypes to assess variability in a populationCreate sequences for phylogenies to determine taxonomic relationshipsConduct forensic investigationsTechnique: (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.
4 Non-examples PCR is NOT used to: Amplify RNA or proteins Construct genomic or cDNA librariesMake monoclonal antibodiesConduct stem cell researchTechnique: If RNA is first reverse transcribed to create cDNA, PCR can be performed on the cDNA.
5 Quick Quiz The purpose of PCR is to: make copies of a specific region of DNAidentify the presence of particular genesmake sufficient genetic material for future molecular workall of the above
6 Thermal Cycler Steps Denature double-stranded DNA Anneal primers to single-stranded DNAExtend primers,yielding new double-stranded DNATechnique: 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: Repeatsteps 1 through 3 ( times)
8 Quick QuizA thermocycler protocol is comprised of which series of steps?Decontamination, amplification, excisionAnnealing, polymerization, coolingTranscription, translation, expressionDenaturing, annealing, extension
9 Quick QuizPCR amplifies molecular products in a(n) __________ fashion:LogarithmicExponentialLinearRandom
10 Quick Quiz Primers: Stabilize double stranded DNA Are enzymes that catalyze the copying processAre short single-stranded DNA fragmentsAre long strings of nucleotides
11 Laboratory PCR Steps (Steps completed by researcher) Create Master Mix of reagentsand aliquot into tubesAdd DNA template(s)Program thermal cycler,load with tubes and startRemove tubes andanalyze results
12 Roles of PCR Reagents GoTaq® PCR Mix Taq polymerase Enzyme that extends growing DNA strand complementary to DNA templateMgCl2Provides ions needed for enzyme reactiondNTP’sNucleotides (Adenine, Cytosine, Guanine, Thymine) building blocks for new DNA strandsBufferMaintains optimal pH for enzymeGreen loading dyeAdds color and viscosity for future gel loading
13 Roles of PCR Reagents Primers DNA template Anneal to single-stranded DNA templateProvide initiation site for extension of new DNAForward primerAnneals to DNA anti-sense strandReverse primerAnneals to DNA sense strandDNA templateIn this case, the product of our DNA extraction
14 Quick Quiz Which of the following reagents is NOT in a master mix? MgCl2Template DNAH2OdNTPs
15 Quick QuizIf you forgot to add one of your primers your resultant gel will probably haveNo bandsA smearA band of the wrong sizeMany bands
16 Setting Up the Reaction PCR reagent volumes vary by experimentAn example ITS amplification in 25 ul final volume is..ReagentReagent concentrationFinal concentrationVolume/ reaction# of reactions= 10GoTaq ® Green PCR mix2X1X12.5 ul125 ulITS forward primer10 M0.3 uM0.75 ul7.5 ulITS reverse primer10 uMWater--1.0 ul10 ulTotal Master Mix15.0 ul150 ulTechinique: 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.)
17 Considerations Contamination can easily lead to erroneous results Avoid contaminating with DNA or PCR product…DNA stocks, PCR reagentsGloves, tips, pipetters, benchesCarefully measure reagent quantitiesUse appropriate cycling conditionsTechnique: When attempting an amplification new to you, find a previous study of a similar organism or tissue and match conditions.
19 Resources http://www.dnai.org/text/mediashowcase/index2.html?id=582 Promega PCR Protocols & ReferencePromega GoTaq Kit HandbookAdditional photos from Cornell University
20 California State Chemistry Standards Grade 85a. Reactant atoms and molecules interact to form products with different chemical properties6c. Living organisms have many different kinds of molecules…Grades 9-122.a. Atoms combine to form molecules by sharing electrons2b. Chemical bonds between many large biological molecules are covalent2c. Salt crystals are repeating patterns of positive and negative ions held together by electrostatic attraction6. Solutions are homogenous mixtures of two or more substances8. Chemical reaction rates depend on factors that influence the frequency of collision of reactant molecules
21 California State Biology Standards Grade 72e. DNA is the genetic material of living organisms and is located in the chromosomes of each cell3a. Genetic variation and environmental factors are causes of evolution and diversity of organismsGrades 9-121b. Enzymes are proteins that catalyze biochemical reactions without altering the reaction equilibrium1d. Molecular biology outlines the flow of information from transcription of RNA to translation of proteins1h. Most macromolecules in cells and organisms are synthesized from a small collection of simple precursors2. Mutation and sexual reproduction lead to genetic variation in a population4. Genes are a set of instructions encoded in DNA…5a. General structure and function of DNA, RNA and proteins5b. Base-paring rules, copying of DNA, replication, transcription5d. Basic DNA techonolgy
22 California State Investigation and Experimentation Standards Grades 7b. Select and use appropriate tools and technology to perform tests, collect data, analyze relationships, and display dataGrade 8a. Plan and conduct a scientific investigation to test a hypothesisGrades 9-12a. Select and use appropriate tools and technology to perform tests, collect data, analyze relationships, and display datal. 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 ScienceContent Standard E: Science and Technology