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

2. 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
Technique: For some applications, PCR and gel electrophoresis are the only steps required to perform analysis. For example, species in an unknown sample can be distinguished by banding patterns observed on a gel if the target amplified in PCR was a substantially different length for each species (typically 50 bp difference or greater).
Diagram by Andy Vierstraete 1999

3. 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
Technique: (1) Multiplex PCR uses more than one set of primers to amplify multiple targets in the same reaction. (2) Quantitative or Real-Time PCR uses fluorescent markers to track the number of copies of target sequence produced in each cycle. With careful calibration, the quantity of DNA present in the original sample can be extrapolated from the number of copies yielded.

4. Non-examples PCR is NOT used to: Amplify RNA or proteins
Construct genomic or cDNA libraries
Make monoclonal antibodies
Conduct stem cell research
Technique: If RNA is first reverse transcribed to create cDNA, PCR can be performed on the cDNA.

5. 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

6. Thermal Cycler Steps Denature double-stranded DNA Anneal
primers to single-stranded DNA
Extend primers,
yielding new double-stranded DNA
Technique: Settings on the thermal cycler can be adjusted for reaction volume and number of cycles, as well as time and temperature of each of the following steps: (1) initial denaturation; (2) cyclic denaturation; (3) cyclic annealing; (4) cyclic extension; (5) final extension; and (6) post-reaction incubation. The total reaction will generally take 1 ½ to 3 hours.
Cycling: Repeat
steps 1 through 3 ( times)

7. PCR Animation--3D

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

9. Quick Quiz
PCR amplifies molecular products in a(n) __________ fashion:
Logarithmic
Exponential
Linear
Random

10. 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

11. 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

12. 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

13. Roles of PCR Reagents Primers DNA template
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

14. Quick Quiz Which of the following reagents is NOT in a master mix?
MgCl2
Template DNA
H2O
dNTPs

15. 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

16. 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
Water --
1.0 ul
10 ul
Total Master Mix
15.0 ul
150 ul
Techinique: Most reactions vary in size from 10 ul to 50 ul. Larger reactions are considered to be more robust because a larger quantity of each reagent is present. (Note that larger reactions increase the cost.)

17. 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
Technique: When attempting an amplification new to you, find a previous study of a similar organism or tissue and match conditions.

18. END

19. Resources http://www.dnai.org/text/mediashowcase/index2.html?id=582
Promega PCR Protocols & Reference
Promega GoTaq Kit Handbook
Additional photos from Cornell University

20. 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

21. 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

22. 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

23. National Standards Grades 6-12 Content Standard A: Science as Inquiry
Content Standard C: Life Science
Content Standard E: Science and Technology