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

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

PCR Animation--3D


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


PCR amplifies molecular products in a(n) __________ fashion:

A. Logarithmic

B. Exponential

C. Linear

D. Random


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


Which of the following reagents is NOT in a master mix?

A. MgCl2B. Template DNA

C. H2O

D. dNTPs


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

END

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

National Standards National Standards

Grades 6-12

Content Standard A: Science as Inquiry

Content Standard C: Life Science

Content Standard E: Science and Technology

Documents

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