intro to pcr the polymerase chain reaction photo courtesy of fisher scientific
TRANSCRIPT
Intro to PCRIntro to PCR The Polymerase Chain ReactionThe Polymerase Chain Reaction
Photo courtesy of Fisher Scientific
DefinitionDefinitionPolymerase 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:
SequencingRestriction digestionBacterial cloning
Diagram by Andy Vierstraete 1999
Application ExamplesApplication Examples
PCR is commonly used to…
Identify speciesIdentify alleles/genotypes to assess variability in a
populationCreate sequences for phylogenies to determine
taxonomic relationshipsConduct forensic investigations
Non-examplesNon-examples
PCR is NOT used to:
Amplify RNA or proteinsConstruct genomic or cDNA librariesMake monoclonal antibodiesConduct stem cell research
Quick QuizQuick Quiz
The purpose of PCR is to:
A. make copies of a specific region of DNA
B. identify the presence of particular genes
C. make sufficient genetic material for future molecular work
D. all of the above
Thermal Cycler StepsThermal Cycler Steps
Cycling: Repeat steps 1 through 3 (20 - 40 times)
Extend primers, yielding new double-stranded DNA
Anneal primers to single-stranded DNA
Denature double-stranded DNA
Quick QuizQuick Quiz
A thermocycler protocol is comprised of which series of steps?
A. Decontamination, amplification, excision
B. Annealing, polymerization, cooling
C. Transcription, translation, expression
D. Denaturing, annealing, extension
Quick QuizQuick Quiz
PCR amplifies molecular products in a(n) __________ fashion:
A. Logarithmic
B. Exponential
C. Linear
D. Random
Quick QuizQuick Quiz
Primers:
A. Stabilize double stranded DNA
B. Are enzymes that catalyze the copying process
C. Are short single-stranded DNA fragments
D. Are long strings of nucleotides
Laboratory PCR StepsLaboratory PCR Steps(Steps completed by researcher)(Steps completed by researcher)
Remove tubes and analyze results
Program thermal cycler, load with tubes and start
Add DNA template(s)
Create Master Mix of reagents and aliquot into tubes
Roles of PCR ReagentsRoles 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’sNucleotides (Adenine, Cytosine, Guanine, Thymine)
building blocks for new DNA strands
Buffer Maintains optimal pH for enzyme
Green loading dyeAdds color and viscosity for future gel loading
Roles of PCR ReagentsRoles of PCR Reagents
PrimersAnneal to single-stranded DNA template
Provide initiation site for extension of new DNA
Forward primerAnneals to DNA anti-sense strand
Reverse primerAnneals to DNA sense strand
DNA templateIn this case, the product of our DNA extraction
Quick QuizQuick Quiz
Which of the following reagents is NOT in a master mix?
A. MgCl2B. Template DNA
C. H2O
D. dNTPs
Quick QuizQuick Quiz
If you forgot to add one of your primers your resultant gel will probably have
A. No bands
B. A smear
C. A band of the wrong size
D. Many bands
Setting Up the ReactionSetting Up the Reaction PCR reagent volumes vary by experiment
An example ITS amplification in 25 ul final volume is..
ReagentReagent
concentrationFinal
concentrationVolume/ 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 0.3 uM 0.75 ul 7.5 ul
Water-- -- 1.0 ul 10 ul
Total Master Mix
-- -- 15.0 ul 150 ul
ConsiderationsConsiderations
Contamination can easily lead to erroneous results
Avoid contaminating with DNA or PCR product…
DNA stocks, PCR reagentsGloves, tips, pipetters, benches
Carefully measure reagent quantities
Use appropriate cycling conditions
ResourcesResources http://www.dnai.org/text/mediashowcase/
index2.html?id=582
Promega PCR Protocols & Referencehttp://www.promega.com/guides/pcr_guide/
Promega GoTaq Kit Handbook http://www.promega.com/tbs/9pim712/_row/9pim7
12_row.pdf
Additional photos from Cornell Universityhttp://www.igd.cornell.edu/MolecularMarkers/PCR
%20basics.pdf
California State Chemistry Standards 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 electrons2b. Chemical bonds between many large biological molecules are covalent2c. 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 StandardsCalifornia State Biology StandardsGrade 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 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 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 proteins5b. Base-paring rules, copying of DNA, replication, transcription5d. Basic DNA techonolgy
California State California State Investigation and ExperimentationInvestigation and Experimentation
StandardsStandards
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