Biol 2281, Spring 2022E7: PCR Introduction, Procedure and Report
Experiment 7: The Polymerase Chain Reaction (PCR) of
human mtDNA
Objectives: At the end of the exercise, you should be able to
1. understand the concepts and principles of polymerase chain reaction.
2. understand and describe the major characteristics of mitochondrial DNA and single
nucleotide polymorphism of the control region sequences.
3. demonstrate laboratory techniques used in DNA isolation, PCR reaction and
visualization of PCR product by DNA agarose gel electrophoresis.
Introduction
1. Polymerase Chain Reaction – Xeroxing DNA
Copyright © 1994-2006 by Access Excellence @ the National Health Museum. Permission to use
this material is granted for educational use. See website at
http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.html.
Who would have thought a bacterium hanging out in a hot spring in Yellowstone National Park
would spark a revolutionary new laboratory technique? The polymerase chain reaction (PCR),
now widely used in research laboratories and doctor’s offices, relies on the ability of DNA-copying
enzymes to remain stable at high temperatures. No problem for Thermus aquaticus, the sultry
bacterium from Yellowstone that now helps scientists produce millions of copies of a single DNA
segment in a matter of hours.
In nature, most organisms copy their DNA in the same way. The PCR mimics this process, only it
does it in a test tube. When any cell divides, enzymes called polymerases make a copy of all the
DNA in each chromosome. The first step in this process is to “unzip” the two DNA chains of the
double helix. As the two strands separate, DNA polymerase makes a copy using each strand as a
template.
The four nucleotide bases, the building blocks of every piece of DNA, are represented by the
letters A, C, G, and T, which stand for their chemical names: adenine, cytosine, guanine, and
thymine. The A on one strand always pairs with the T on the other, whereas C always pairs with
G. The two strands are said to be complementary to each other.
To copy DNA, polymerase requires one other component: a primer. DNA polymerases, whether
from humans, bacteria, or viruses, cannot copy a chain of DNA without a short sequence of
nucleotides to “prime” the process, or get it started. So the cell has another enzyme called a
primase that actually makes the first few nucleotides of the copy. This stretch of DNA is called a
primer. Once the primer is made, the polymerase can take over making the rest of the new chain.
A PCR vial contains all the necessary components for DNA duplication: a piece of DNA, large
quantities of the four nucleotides, large quantities of the primer sequence, and DNA polymerase.
The polymerase (in PCR) is the Taq polymerase, named for Thermus aquaticus, from which it
was isolated.
The three parts of the polymerase chain reaction are carried out in the same vial, but at different
temperatures. The first part of the process separates the two DNA chains in the double helix. This
is done simply by heating the vial to 90-95 degrees centigrade for 30 seconds. But the primers
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Biol 2281, Spring 2022
E7: PCR Introduction, Procedure and Report
cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 55-58 degrees
C. At this temperature, the primers bind or “anneal” to the ends of the DNA strands. This takes
about 30 seconds. The final step of the reaction is to make a complete copy of the templates.
Since the Taq polymerase works best at around 75 degrees C (the temperature of the hot springs
where the bacterium was discovered), the temperature of the vial is raised. The Taq polymerase
begins adding nucleotides to the primer and eventually makes a complementary copy of the
template. This completes one PCR cycle.
The three steps in the polymerase chain reaction – the separation of the strands, annealing the
primer to the template, and the synthesis of new strands – take less than two minutes. Each is
carried out in the same vial. At the end of a cycle, each piece of DNA in the vial has been
duplicated. But the cycle can be repeated 30 or more times. Each newly synthesized DNA piece
can act as a new template, so after 30 cycles, 1 billion copies ( 230) of a single piece of DNA can
be produced!
PCR is valuable to researchers because it allows them to multiply unique regions of DNA so they
can be detected in large genomes. Thanks to this procedure, one can make billions of copies of a
single DNA molecule even though it is initially present in a mixture containing many different DNA
molecules.
There are some limitations of PCR reactions: 1) Contamination of the DNA sample from other
sources can cause problems. 2) Taq DNA polymerase has no proof-reading function, leading to a
relatively high mutation rate (1/104); but now thermostable DNA polymerases with proof-reading
activity are available.
2. Human Mitochondrial DNA
The following material was produced by the DNA Dolan Learning Center at Cold Spring Harbor
Laboratory, Long Island, New York. See website:
http://www.geneticorigins.org/geneticorigins/mito/mitoframeset.htm. Permission to use this
material is granted for educational use.
Every human cell has a “second” genome, found in the cell’s energy-generating organelle, the
mitochondrion. In fact, each mitochondrion has several copies of its own genome, and there are
several hundred to several thousand mitochondria per cell. This means that the mitochondrial (mt)
genome is highly amplified. While each cell contains only two copies of a given nuclear gene (one
on each of the paired chromosomes), there are thousands of copies of a given mt gene per cell.
Because of this high copy number, it is possible to obtain a mt DNA type from the equivalent of a
single cell’s worth of mt DNA. Thus, mt DNA is the genetic system of choice in cases where
tissue samples are very old, very small, or badly degraded by heat and humidity.
Under good circumstances – working from fresh cell samples – mt DNA is the easiest human DNA
to amplify by PCR. This experiment examines a 440-nucleotide sequence from the noncoding
region of mt genome. Hand cycling is a realistic alternative to automated thermal cyclers, and the
high yield of amplified product can be visualized in an agarose gel with a variety of stains.
Because each student is amplifying the same region, the gel electrophoresis results will also be
the same for each. However, amplified student samples may be submitted to our Sequencing
Service, which will generate student mt DNA sequences and post the results on our Sequence
Server. Comparison of control region sequences reveals that most people have a unique pattern
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E7: PCR Introduction, Procedure and Report
of single nucleotide polymorphisms (SNPs). These sequence differences, in turn, are the basis
for far-ranging investigations on human DNA diversity and the evolution of hominids.
The entire DNA sequence of the human mt genome – 16,569 nucleotides – was determined in
1981, well in advance of the Human Genome Project. The mt genome contains 37 genes, all of
which are involved in the production of energy and its storage in ATP. Thirteen of these genes
encode proteins involved in oxidative phosphorylation. The remaining genes encode transfer
RNAs (22 genes) and ribosomal RNAs (2 genes) that translate the proteins’ genes within the
mitochondrion. Mammalian mt genes use a slightly different genetic code than nuclear genes,
where UGA = tryptophan, AUA = methionine, and AGA and AGG = stop.
Genes take up the majority of the mt genome. However, a noncoding region of approximately
1,200 nucleotides spans both sides of the arbitrary “0” position of the mt genome and goes by
three confusing terms: control region, D-loop, and hypervariable region. Control region refers to
the fact that this region contains the signals that control RNA and DNA synthesis. A single
promoter on each DNA strand initiates transcription in each direction, and a single origin initiates
replication of each strand. D-loop refers to the early phase of replication, when the first newlysynthesized strand displaces one of the parental strands, forming a “bubble” or loop. The DNA
sequence of the control region is termed hypervariable, because it accumulates point mutations
at approximately 10 times the rate of nuclear DNA.
3. The puReTag Ready-To-Go PCR Beads from “GE HEALTHCARE”.
Each bead is a premixed, predispensed reaction for PCR featuring PuReTaq DNA polymerase,
when suspended in a final volume of 25 μl, will contain the following:
•
•
2.5 units of puReTaq DNA Polymerase
10 mM Tris-HCl, pH 9.0 at room temperature
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Biol 2281, Spring 2022
E7: PCR Introduction, Procedure and Report
•
•
•
50 mM KCl
1.5 mM MgCl2
200 μM of each dNTPs
Day 1
Material: per student
Swab
Marker pen
One microcentrifuge tube labeled ‘S’ with 500ul sterile water
Three empty microcentrifuge tubes and tube rack.
10% Chelex®, 100 µl (return colored tube to ice after use)
Micropipettes (P200, and P20)
One yellow tip box
Primer/loading buffer mix (20 µl): provided by the TA
Ready-to-Go PCR Bead (0.2 ml tube): provided by the TA
Shared items
Microcentrifuge
Hot plate for boiling water
Thermal cycler
Extra microcentrifuge tube to use as balance if needed
The following procedure was based on information produced by the DNA Dolan Learning Center
at Cold Spring Harbor Laboratory, Long Island, New York. Parts of the procedure were modified
to suit the experimental equipment and supplies available in the UG lab at UT Dallas. Permission
to use this material is granted for educational use.
Part I: Total DNA Isolation by cheek swab
initials #1
initials #2
WASTE
1. Label tube S (the microcentrifuge tube with 500ul water) with your initials.
2. Label the cap of the other microcentrifuge tubes with your initials 1, initials 2,
WASTE respectively.
3. Take the sterile swab and swab the inside of each cheek for 10 seconds each (total of
20 seconds). You may go outside the lab to collect your sample if you wish.
4. Dissolve your sample from the swab into tube S by inserting the swab into the water
and twisting it for 10 seconds.
5. Place tube S in the microcentrifuge (do not forget to use a balance tube or spin with
another student). Spin tube S in the microcentrifuge for 2 minutes.
6. Carefully pour off supernatant into your waste tube. Be careful not to disturb the cell
pellet at the bottom of the tube. A small amount (0.1 ml) of water and the pellet should
remain in tube S. Discard your swab and your waste tube in the large biohazard bin.
7. Resuspend and mix cells in tube S (pellet) in remaining water (0.1 ml) by pipetting up
and down using the P200 micropipette.
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E7: PCR Introduction, Procedure and Report
8. Set pipet at 40 µl. Transfer 40 µl of cell suspension from Tube S to Tube 1.
9. Set pipet at 100 µl, attach one clean tip.
10. Take the microcentrifuge tube (colored tube) containing Chelex from the ice box. Tap
the Chelex tube (or use vortexer) to mix and resuspend the white particles thoroughly.
Before the white particles settle down, quickly pipet 100 µl of Chelex into tube 1
(containing 40ul of your cell suspension). Shake well to mix.
11. Return the colored Chelex tube to the ice right away.
12. Close the cap. Boil cell sample in tube 1 for 10 minutes in a boiling water bath. (After
the water comes to a boil, reset the hot plate temperature setting to between 6 and 7).
You may obtain PCR Bead in a 0.2 ml tube (reaction tube) and 20 µl of the
primer/loading buffer mix from TA during the 10 minutes. (See step 1 in Part II).
13. After boiling, place tube 1 in a balanced configuration with other tubes, spin for 2
minutes.
14. Add a new pipette tip to the P20 and transfer 20 µl of supernatant (top liquid layer
containing the DNA) to a clean 1.5 ml tube (Tube 2). Avoid cell debris and Chelex
beads.
Note:
1) From Bio-rad: “Chelex 100 molecular biology grade resin is a highly pure, pipettable,
nuclease and ligase inhibitor-free chelating resin specifically designed and certified for
extraction of PCR-ready template DNA. Chelex 100 molecular biology grade resin
accommodates the stringency required for a PCR-quality product by ensuring the
complete removal of PCR inhibitors (contaminating metal ions that catalyze the
digestion of DNA).”
2) Boiling the cell mixture serves to lyse the cell to release the DNA and at the same
time, inactivate cellular proteins that may interfere with the PCR reaction.
Part II: DNA Amplification by PCR
1. Obtain a PCR reaction tube (containing a small Ready-To-Go PCR Bead) from TA.
(TA supervision) Use a micropipette with a fresh tip to add 20 µl of the appropriate
primer/loading buffer mix to small PCR reaction tube. Tap the tube to dissolve bead. The
Ready-To-Go PCR Bead is a patented product that contains the Taq polymerase.
2. Use P20 micropipette and fresh tip to add 5.0 µl of human DNA (Tube 2 from Part I) to
small PCR reaction tube, and tap to mix.
Label the cap of small PCR reaction tube with your sign-in number and section
number. The volume of your sample in PCR tube should be ______ µl now.
3. Place your small PCR reaction tube in the ice box next to the PCR machine.
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E7: PCR Introduction, Procedure and Report
Cleaning Up:
1. Dispose the used swabs, tips, microcentrifuge tubes in the big biohazard trashcan.
2. Place tip box, tube rack and micropipette to original containers.
Store your small PCR reaction tube on ice until ready to amplify according to the
following profile. Program thermal cycler for 30 cycles according to the following cycle
profiles. At the end of the program, the amplified DNAs will be held at 4°C. Your
samples will be transferred to a freezer until next lab to be analyzed by gel
electrophoresis.
Step 1: Denaturation: 30 sec at 940C
§
The reaction mix is heated to about 940C. At this temperature, the dsDNA
dissociate into single strands
Step 2: Annealing of Primers: 30 sec at 580C
§
Next, the solution is allowed to cool to about 580C. As it cools, the single strands of
DNA re-associate into double strands according to sequence complementarity.
However, because of the large excess of primer, each strand of the DNA template
base-pairs with a complimentary primer, leaving the rest of the DNA template
single stranded.
Step 3: Primer Extension: 30 sec at 720C
§
Using the primers, the heat-stable DNA polymerase (Taq polymerase) copies the
rest of the DNA template as if it were replicating DNA. When it is done, the primer
has been lengthened into a complimentary copy of the entire single-stranded
fragment. Because both DNA template strands are replicated, there are now two
copies of the original fragment.
Step 4: Repeat the above three steps 30 times
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Biol 2281, Spring 2022
E7: PCR Introduction, Procedure and Report
DAY 2
Part III: DNA Analysis by Gel Electrophoresis
1.
2.
3.
4.
5.
6.
0.5X TBE buffer
1µg/ml ethidium bromide (provided by the TA)
100 bp DNA molecular size marker
agarose
micropipette (P20) and yellow tip box
electrophoresis equipment and power supply
Procedure
1. (TA) Prepare a 1.5% agarose gel with a total volume of 50 ml in 0.5X TBE. Heat it to
melt the agarose; How much agarose to weigh? _________________ grams.
2. TA will add 1 µl of EB solution (10mg/ml) to your melted agarose gel BEFORE pouring
to the gel tray.
3. Use a micropipette with a fresh tip to load 10µl PCR product into your assigned well of
the gel. Expel any air from the tip before loading, and be careful not to push the tip of the
pipet through the bottom of the sample well.
4. (TA) Load 5 µl of the 100 bp-DNA size marker (ladder) into one lane of the gel.
5. Electrophorese at 130 volts for 35-40 minutes. Adequate separation will have occurred
when the cresol red dye front has moved at least 50 mm from the wells.
6. Check the map on page 3, based on the locations of the two primers to predict the size
of the PCR product. The size is predicted to be ___________bp. Examine the gel using
UV lamp; estimate the size of your PCR product by comparing to the 100 bp-DNA size
marker (ladder).
Part IV: (Optional) DNA Sequencing by Dideoxy Chain Termination
The sequence of amplified mt DNA samples could be analyzed using cycle sequencing.
Animation of cycle sequencing can be viewed at http://www.geneticorigins.org/mito/media.html.
You can access sequences of amplified mt DNA samples by using a tool “Sequence Server.” at
http://www.bioserver.org/sequences/.
After logging in (click “ENTER”) to Sequence Server, you should click the “Manage Groups”
button at the top of the screen. This will open the Manage Groups window. In this window,
choose “Classes” from the popup menu “Sequence Sources” on the upper-right. A new screen
will appear, and you will be able to see many class’s name in the list presented. To select any
class, click on the checkbox next to a class, and click the OK button. This will put specific class
on your worksheet.
To compare sequences, you need to have more than one sequence on the worksheet. To add
a sequence, select the desired sequence from the popup menu at the bottom of your class. Do
this for as many sequences as you want. Then, click the checkbox for each sequence you wish
to include in a comparison, and then click on the “Compare” button. Sequence Server will open
a new window showing the results of your analysis. You can include the sequence of
Neandertal mtDNA by choosing prehistoric DNA from “sequence sources”.
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Biol 2281, Spring 2022
E7: PCR Introduction, Procedure and Report
Post Lab Report (20 pts total): Include title page (0.5 pt) and questions (0.5 pt) in
your report
1. Presentation of the picture of the gel, with proper labeling of each lane, wells, the sizes
of the marker bands, the anode and cathode (2 points). (photos posted on eLearning)
2. How did you predict the size of the PCR product (Human mitochondrial genome map on
page 3)? What is the estimated size of your PCR product when you compare it to the
DNA ladder? Why was the 100 base-pair ladder used instead of 500 bp ladder for this
exercise? (3 points)
3. Each round of PCR doubles the number of DNA molecules present in the reaction. How
many molecules can be generated from one of your DNA template molecules after 30
cycles? (1 point)
4. You amplified a segment of your own mitochondrial DNA. Do you expect the sequences
of your PCR product vary from those of your classmates? Why or Why not? (2 points)
5. What does the term “denaturation” mean in relation to DNA? (2 pts)
6. What does the term “annealing” mean in relation to DNA? What kinds of chemical bonds
are formed when two strands of DNA anneal? (2 points)
7. The process of PCR is dependent upon the use of certain DNA polymerases.
a)
Why are they special and where do they come from? (2 points)
b)
Why does the PCR reaction catalyzed by the Taq DNA polymerase have a
higher mutation rate than the normal DNA replication process found in living cells?
(1pt)
8. What is the basis of separation of DNA molecules in the agarose gel that you run in this
experiment? What are the electrical charges of the DNA molecules? (2 pts)
9. What reagent is added to the 0.5X TBE buffer to allow visualization of the DNA bands in
the agarose gel? Describe how you can see the DNA after the gel running is stopped. (2
pts)
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