Intro to PCR The Polymerase Chain Reaction

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

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Non-examples

• PCR is NOT used to
• Amplify RNA or proteins
• Construct genomic or cDNA libraries
• Make monoclonal antibodies
• Conduct stem cell research

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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

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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)
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• PCR Animation--3D

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Quick Quiz

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

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Quick Quiz

• PCR amplifies molecular products in a(n)
  __________ fashion
• Logarithmic
• Exponential
• Linear
• Random

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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

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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
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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
• dNTPs
• 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

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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

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Quick Quiz

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

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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

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Setting Up the Reaction

• PCR reagent volumes vary by experiment
• An example ITS amplification in 25 ul final
  volume is..

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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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• END

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Resources

• http//www.dnai.org/text/mediashowcase/index2.html
  ?id582
• Promega PCR Protocols Reference
• http//www.promega.com/guides/pcr_guide/
• Promega GoTaq Kit Handbook
• http//www.promega.com/tbs/9pim712/_row/9pim712_ro
  w.pdf
• Additional photos from Cornell University
• http//www.igd.cornell.edu/MolecularMarkers/PCR20
  basics.pdf

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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

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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

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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

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National Standards

• Grades 6-12
• Content Standard A Science as Inquiry
•  
• Content Standard C Life Science
• Content Standard E Science and Technology