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

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

Non-examples

PCR is NOT used to:

Amplify RNA or proteins Construct genomic or cDNA libraries

Make monoclonal antibodies

Conduct stem cell research

Quick Quiz
The purpose of PCR is to:
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
A.

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)

PCR Animation--3D

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

Quick Quiz
PCR amplifies molecular products in a(n) __________ fashion:
A. B. C. D.

Logarithmic Exponential Linear Random

Quick Quiz
Primers:
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
A.

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

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

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

Quick Quiz
Which of the following reagents is NOT in a master mix?
A. B. C. D.

MgCl2 Template DNA H 2O dNTPs

Quick Quiz
If you forgot to add one of your primers your resultant gel will probably have
A. B. C. D.

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 concentration 2X Final concentration 1X Volume/ reaction 12.5 ul # of reactions = 10 125 ul

Reagent GoTaq Green PCR mix ITS forward primer ITS reverse primer Water Total Master Mix

10 M
10 uM ---

0.3 uM
0.3 uM ---

0.75 ul
0.75 ul 1.0 ul 15.0 ul

7.5 ul
7.5 ul 10 ul 150 ul

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

END

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/9pim7 12_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