Techniques: Polymerase Chain Reaction
Background
The polymerase chain reaction (PCR) is used to make many copies of a defined segment
of a DNA molecule. Here’s how it works. First, you decide what DNA segment you wish
to duplicate, or amplify. Then you obtain two short single-stranded DNA molecules that
are complementary to the very ends of the segment. These two short molecules must have
specific characteristics. Each of the single-stranded molecules must have a base sequence
that is complementary to only one strand of the target DNA, and each one must
be complementary to only one end of the segment. Furthermore, if you imagine these
short molecules base paired to the complementary regions in the target DNA molecule,
their 3′ ends would point toward each other. These short, single-stranded molecules are
the primers for PCR (see the figure below).
To begin the chain reaction, a large number of primers is mixed with the target molecule
in a test tube containing DNA polymerase enzyme, buffer and many deoxynucleoside
triphosphates. This mixture is heated to almost boiling, so that the base pairs holding the
two strands of the parental molecule separate, or denature (Denaturation in the figure).
Next, the mixture is allowed to cool. Ordinarily, the two strands of the target DNA
molecule would eventually line up and re-form their base pairs. However, there are so
many molecules of primers in the mixture that the short primers find their complementary
sites on the target strands before the two target strands can line up correctly for base
pairing. Therefore a primer molecule base pairs, or hybridizes, to each of the target
strands (Hybridization in the figure).
Now DNA polymerase enzyme adds deoxynucleotides to the 3′ end of each primer, using
the bases on the target strand as a template. New complementary strands are synthesized,
and the 5′ end of each is formed by a primer (DNA Synthesis in the figure). In this
manner, two double-stranded molecules are formed where before there was only the
single target molecule.
This process of denaturation, hybridization, and DNA synthesis is repeated over and
over. Each time, the number of DNA molecules in the mixture is doubled. The
overwhelming majority of the newly synthesized molecules extend exactly from one
primer sequence to the other. So by choosing the primers, a scientist controls which
segment of the target molecule is amplified. We have chosen primers which have a
specific sequence to amplify the green fluorescent protein (GFP) gene. In addition, the
5’ terminal ends of both primers possess a HindIII restriction endonuclease site, which
we will exploit during the cloning of our amplified gene into an expression vector.
Amplifying the Green Fluorescent Protein gene
The Green Fluorescent Protein (GFP) gene is from a bioluminescent jellyfish, Aequorea
victoria. These jellyfish emit a green glow from the edges of their belllike structures. This
glow is easily seen in the coastal waters inhabited by the jellyfish. We do not know the
biological significance of this luminescence.
The GFP protein glows by itself; it is auto-fluorescent in the presence of ultraviolet light.
Because of this self-glowing feature, GFP has become widely used in research as a
reporter molecule. A reporter molecule is one protein (such as the gene for GFP) linked
to the protein that you are actually interested in studying. Then you follow what your
protein is doing by locating it with the reporter molecule. For instance, if you wanted to
know whether a particular gene (gene X) was involved in the formation of blood vessels,
you could link (or fuse) gene X to the GFP gene. Then, instead of making protein X, the
cells would make a protein that was X plus GFP. The type of protein that results from
linking the sequences for two different genes together is known as a fusion protein. If the
blood vessels began glowing with GFP, it would be a clue that protein X was usually
present and a sign that X might indeed be involved in blood vessel formation.
Purpose
The purpose of this exercise is to amplify a target DNA sequence from the genome of
Aequorea victoria, to subsequently clone the gene into an expression vector.
Materials per team
PCR machine
PCR Tube and PCR Tray
10 M PCR primers (2 total)
Mineral Oil
Forceps
P20, P200 Pipetman
10X PCR reaction buffer
A. victoria DNA (17 ng/L)
4oC refrigerator
Microcentrifuge Tube Rack
Pipetman Tips
8 mM dNTP mix
Taq Polymerase
Latex Gloves
Procedures
1. REMEMBER TO USE ASEPTIC TECHNIQUE! THIS IS FOR REAL!
2. Make the following mixture in a PCR tube with pipetmen (100 L total volume):
10 L 10X PCR reaction buffer
10 L 8 mM neutralized dNTP mix solution (in 1.5 mM Mg+2 solution)
10 L 10 M ‘sense’ PCR primer (100 picomoles)
10 L 10 M ‘antisense’ PCR primer (100 picomoles)
59 L template DNA (total of 1 g of chromosomal DNA in water)
1 L Thermus aquaticus (Taq) DNA polymerase (2 Units of enzyme)
3. Mix the contents of the tube with a P200 pipetman (set the pipetman to 100 L and
mix by pipeting the contents up-and-down twice).
4. Overlay the reaction mixture with mineral oil (just enough to cover the reaction).
5. Place the tube in the PCR machine, set the machine to the following parameters:
Step 1: 2.0 minutes, 94oC (denaturing step: DNA template strands separate)
Step 2: 1.5 minutes, 55oC (hybridizing step: Primers anneal to the DNA template)
Step 3: 1.0 minute, 72oC (synthesis step: Complementary DNA is produced)
Step 4: Repeat cycles 1 through 3 (25 times) (this is the cycling component)
Step 4: Infinite time (set as ‘hold’), 4oC (keeps sample cold until stored)
6. Run the PCR machine (it will cycle automatically until the run is completed).
7. Your instructor will store the samples in the refrigerator (4oC) until next period.
8. Complete the attached Worksheet.
WORKSHEET
Polymerase Chain Reaction (PCR)
1. What is the goal of PCR?
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2. Why are two sequence-specific primers necessary for PCR amplification to work?
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3. Why is it necessary to denature the target DNA before producing new DNA?
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4. Why is mineral oil employed as an overlay during the PCR procedure?
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5. During the hybridizing step, why does the template DNA not re-anneal to each other?
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6. You have produced an excellent, full-length, high-quality product of amplified DNA.
Unfortunately, it is limiting in quantity. What would you do to increase the quantity
of amplified DNA? How would you modify the PCR procedure?
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7. You are amplifying a sequence of DNA which is high in GC content (3 Hydrogen
bonds). How would you modify the PCR procedure from the one you were using for
DNA high in AT content (2 Hydrogen bonds)?
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8. Can you propose a reason Jellyfish have Green Fluorescent Protein (and don’t tell me
those that didn’t express it didn’t reproduce – this is correct, however)?
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9. You want to prove the cellular localization of a protein you propose is involved in
DNA repair. How would you use GFP to accomplish this?
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