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
 dNTP’s
 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
H2O
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
Reagent
concentration
Final
concentration
Volume/
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
--
--
1.0 ul
10 ul
--
--
15.0 ul
150 ul
Water
Total Master
Mix
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