UNIT 7 – DNA AND MOLECULAR BIOLOGY
PRE-LAB ASSIGNMENT
NAME _____________________
Review Sections 14.3, 14.5, 15.3, 16.1, and Chapter 17 in your textbook as well as relevant
lecture notes and the remainder of this laboratory handout. Answer the following questions.
-Explain what is meant by the 5’-to-3’ strand and 3’-to-5’ strand of DNA. What is the origin of the
3’ and 5’ in these terms?
-What is a “primer” and what exactly happens when it “anneals” to a DNA sequence?
-What is the difference between a forward primer and reverse primer?
-What does a polymerase do?
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- What does a restriction endonuclease do?
-Upon completing your gel electrophoresis you will be able to observe distinct banding patterns
between oyster mushroom and portabella mushroom. You will also be able to determine the size of
the DNA fragments that formed the electrophoresis bands. How, exactly, will you determine the size
of these DNA fragments?
-At what point in today’s procedure will you be certain to where gloves?
-On page 7-12 you will be asked to state a predictive, testable hypothesis. State that hypothesis here.
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UNIT 7 – DNA AND MOLECULAR BIOLOGY
Objectives:
The objectives of this laboratory are to:
 illustrate the flow of genetic information in biological systems – that is, DNA is transcribed
into RNA which is translated into protein. (Figure 7.1);
 review the mechanisms of DNA replication; and
 explore practical applications of molecular biology techniques.
For important background information on this laboratory, you should study Section 17.3 (DNA
Analysis) of your textbook.
You will conduct two experiments during this laboratory. In the first experiment you will identify
two species of fungi using a technique that analyzes differences in DNA fragment lengths beginning
from a common starting point (this is called a restriction fragment length polymorphism analysis or
RFLP). The second experiment will demonstrate that the genetic control of enzyme synthesis
(translation) by the bacterium Escherichia coli can actually be influenced by environmental factors.
In order to carry out these experiments you will be introduced to a number of modern molecular
techniques including PCR, restriction digest, and gel electrophoresis.
Figure 7.1: The flow of genetic information in biological systems.
Experiment 1: Restriction fragment length polymorphism analysis of
fungal DNA
Imagine that you are working for a very forgetful mycology professor. Your professor has isolated
DNA from two fungal species, Agaricus bisporus (portabella mushroom) and Pleurotus ostreatus
(oyster mushroom), which were collected in the field, but due to improper labeling, has mixed up the
vials containing the extracted DNA. She has instructed you to perform a restriction fragment length
polymorphism analysis (RFLP) to determine which vial belongs to which species. An RFLP is
actually a process made up of several steps. The first step involves the replication of a specific
region of DNA in order to produce sufficient quantities of DNA for analysis. This step uses a
polymerase chain reaction (PCR) to replicate the target DNA region. The technology to perform
PCR is based upon our knowledge of the natural DNA replication process in cells. The second step
is a digest that uses a restriction enzyme to cut the replicated DNA at specific locations. In the third
and final step gel electrophoresis is performed to determine the size of the DNA fragments that
resulted from the restriction enzyme digests on the DNA of the two fungal species.
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PCR (Polymerase Chain Reaction)
Polymerase chain reaction is a method that amplifies (copies) small segments of DNA. PCR
technology mimics cellular DNA replication in many ways and even utilizes a polymerase
enzyme. In this experiment, you will replicate a segment of fungal DNA called the internal
transcribed spacer (ITS) region. This region is highly variable among species and it is flanked
by highly conserved regions. The high sequence variability in the ITS allows us to distinguish
between species, while the conserved regions (regions of low or no variation) flanking the ITS
permit us to use the same primers for all fungi. As a result, the ITS region functions as a
barcode for identification of fungal species. The goal of this portion of the experiment is to
amplify this target region of DNA using PCR.
The necessary biochemical components for PCR are provided in a PCR Master Mix. This
master mix will contain all the components required to replicate DNA. The first component,
the primer, is a small, complementary sequence of DNA that binds (“anneals”) to a specific
region in the genome and serves as the starting point at which other enzymes will begin
replicating the remaining DNA sequence. Primers are always used in pairs, one complementary
to the 5’-to-3’ strand of DNA and the other complementary to the 3’-to-5’ strand of DNA.
Typically these are referred to as forward and reverse primers (Figure 7.2). The primer pair that
we will use in this experiment has been designed to bind to the sequences that flank our target
region of fungal DNA. Because the regions where the primers bind are conserved between
species, the same primers will work for all fungi. In living cells, the enzyme primase produces
primers that serve to define the segments of DNA to be replicated or transcribed.
Forward Primer ITS1
SSU rDNA
5.8S
LSU rDNA
Reverse Primer ITS4
ITS variable region
Figure 7.2 Forward and reverse primers used in this experiment to amplify the “variable region” of DNA
in the fungal genome.
► Segments of two complementary strands of DNA are depicted in the space below. The 5’-to3’ strand is the top strand and the complementary 3’-to-5’ strand is on the bottom. Design a
forward primer complementary to the bold sequence in the 5’-to-3’ strand, along with a
reverse primer complementary to the bold sequence in the 3’-to-5’ strand. Write in the
nucleotide sequence of the primers immediately adjacent to the bold sequences in a manner
you would expect to see them when they anneal to those sequences. Once the primers
anneal to their complementary strands they will mark the location to begin replicating the
target DNA sequences.
Forward primer here
5’-GAGTCGAGTTCGAAAGCTTCGAGCCTGAGCTTCGATCGTGCTAGCTAGAGC-3’
3’-CTCAGCTCAAGCTTTCGAAGCTCGGACTCGAAGCTAGCACGATCGATCTCG-5’
Target sequence to be replicated
Reverse primer here
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The second component of the master mix is a special DNA polymerase known as Taq
polymerase. DNA polymerase is an enzyme that synthesizes DNA by linking nucleotides (A,
G, C, and T). Taq polymerase is a DNA polymerase that is particularly heat-stable, meaning
that it can function at high temperatures without denaturing (we want this because our PCR will
be running at temperatures up to 95C). The third component of the master mix is free-floating
nucleotides: A, C, T, and G. These nucleotides will be used by Taq polymerase to construct
numerous copies of our sequence of interest. The final component is a buffer that works to hold
an optimal pH in the master mix.
Fungal DNA will be added to the master mix and placed into a machine called a thermal cycler
(Figure 7.3). The thermal cycler works by adjusting the temperature conditions over time. The
PCR process works in three main steps: (1) Denaturation Step, (2) Annealing Step, and (3)
Elongation Step. Each of these steps occurs at a different temperature for a specific amount of
time. The three steps taken together in sequence comprise one PCR cycle.
Figure 7.3. Polymerase Chain Reaction (PCR) techniques are used to amplify DNA sequences. See text
for details (image from Raven et al. 2008).
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(1) In the denaturation step the hydrogen bonds between the nitrogenous bases break,
separating the two strands of DNA. This is accomplished by an increase in temperature to
95C.
► How does an increase in temperature work to break the hydrogen bonds between
complementary nitrogenous bases of DNA?
(2) In the annealing step, the forward and reverse primers bind to their complementary strands
of DNA. This is accomplished by decreasing the temperature to 48C.
(3) Finally, in the elongation step the Taq polymerase binds to the end of the primer sequence
and synthesizes the remaining complementary strand of DNA, utilizing the nucleotides in
the master mix. This is accomplished by increasing the temperature to 72C.
At the end of the first cycle of PCR, two new strands of DNA will be synthesized – one
complementary to the 5’-to-3’ strand and the other complementary to the 3’-to-5’ strand. This
process is repeated for 35 cycles, each cycle exponentially increasing the number of copies of
our target sequence of DNA. Once PCR is completed, we will have millions of copies of our
sequence of interest.
Restriction Fragment Length Polymorphism
The second portion of this experiment will include a restriction digest of the amplified fungal DNA
sequence. Restriction digests utilize naturally occurring enzymes known as restriction
endonucleases. These enzymes work by cleaving DNA at specific locations (cleavage sites). Each
enzyme has a single sequence of DNA that it recognizes and cuts in a specific way. In this
experiment, you will use the restriction endonuclease HaeIII to cut the amplified fungal DNA
samples into fragments.
As stated previously, the region of fungal DNA that was amplified with PCR varies among different
fungal species. Depending on the species, the number and location of cleavage sites for a given
restriction endonuclease will differ based upon the range of unique nucleotide sequences that exist
between the cleavage sites in respective species. Using a single restriction endonuclease to cut our
fungal DNA will produce different sized fragments for our two unknown samples. A restriction
digest with HaeIII using portabella mushroom DNA will produce two distinct fragments, one having
550 base pairs and the other having 220 base pairs. The restriction digest with HaeIII using oyster
mushroom DNA will produce three fragments having 250, 210 and 200 base pairs. If we can
visualize this difference in fragment size, we can distinguish DNA samples from the two fungal
species.
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Gel Electrophoresis
In the final portion of this experiment, you will use gel electrophoresis to
visualize the fragments produced in the restriction digest as bands on an agarose
gel. The gels that you will use are made with ethidium bromide, which is a
chemical that will bind to the fragments of DNA as they migrate down the gel.
When placed under ultraviolet light, the band will glow because of the addition
of ethidium bromide. Extended exposure to ethidium bromide can be hazardous,
so it is important to wear gloves if you handle the electrophoresis gels or buffer.
In addition to your samples, you will also load a molecular weight marker into
one of the lanes of the gel. The molecular weight marker produces a pattern of
bands of known size (Figure 7.4). We can compare the location of our fungal
DNA bands to those in the molecular weight marker and approximate the size of
those fragments of DNA. This will allow you to distinguish the oyster
mushroom from the portabella mushroom.
Figure 7.4 100-bp
molecular weight
markers (Source:
http://www.neb.com/ne
becomm/products/produ
ctN3231.asp)
► You will eventually produce an electrophoresis gel having four lanes. One lane will contain the
molecular weight markers. Another lane will contain oyster mushroom DNA fragments after
exposure to HaeIII. The third lane will contain portabella mushroom DNA fragments after
exposure to HaeIII.
The fourth lane will be discussed below, but for the moment, use your prior understanding of gel
electrophoresis to depict the banding patterns you expect to see in these three lanes. Using the
hypothetical electrophoresis chamber, depicted below, identify the size (# of base pairs) of the
DNA fragments that make up each of the bands in the three lanes. Also, use your understanding
of DNA molecules to properly label the positive and negative poles of the electrophoresis
chamber.
Load here
Molecular weight markers
Oyster mushroom DNA
Portabella mushroom DNA
+ or –
pole?
+ or –
pole?
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► You may have heard that portabella mushrooms are simply a brown variety of the common white
button mushroom. Producers simply let the button mushrooms grow large and brown, and permit
them to open and expose their gills – for this you get to pay 4-5 times more at the grocery store
for portabella mushrooms than for white button mushrooms. In the diagram below, reproduce the
banding patterns from the previous diagram, but now include the banding pattern you would
expect to see if, in fact, white button mushrooms are the same species as portabella mushrooms.
Load here
Molecular weight markers
Oyster mushroom DNA
Portabella mushroom DNA
White button mushroom DNA
+ or –
pole?
+ or –
pole?
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Procedures: PCR and RFLP Analysis of Fungal DNA
1.
Setting up a PCR Reaction
- Obtain 3 PCR tubes (0.5 ml tubes) and label one “Species A”, one “Species B” and the third
“White.” Also write the date and group number on the side of the tube.
- Place the tubes on ice.
- Using a micropipette and a fresh, sterile tip, pipette 15 µl of the PCR Master Mix (containing
the PCR primers, Taq polymerase, and nucleotides) into each of the tubes (Tubes A, B and
White). Return the tubes to the ice bucket.
► Why is it important to keep the PCR components constantly on ice? (Think about optimal
temperature conditions for enzymes.)
- Using a micropipette and a fresh tip, pipette 5 µl of the DNA extract from species A into the
“Species A” PCR tube and place on ice.
- Using a micropipette and a fresh tip, pipette 5 µl of the DNA extract from species B into the
“Species B” PCR tube and place on ice.
- Using a micropipette and a fresh tip, pipette 5 µl of the DNA extract from white button into
the “White” PCR tube and place on ice.
- The tubes will be placed into the thermal cycler by your TA and used by students in the next
lab section.
► What is the function of the thermal cycler?
2.
Setting up a Restriction Digest using HaeIII
- Obtain one “Species A” PCR tube, one “Species B” PCR tube, and one “White” PCR tube
from the completed PCR ice bucket. (*NOTE: These reactions were set-up and completed
by the previous lab section.)
- Obtain three PCR tubes and label one “Species A RD”, label one “Species B RD”, and label
the third “White RD”.
- Using a micropipette and fresh tip, pipette 18 µl of the Restriction Digest Mix (contains
enzyme, buffer and water) into the “Species A RD”, “Species B RD”, and “White RD”
tubes and place on ice.
- Using a micropipette and fresh tip, pipette 10 µl of PCR product from the “Species A” PCR
tube into the “Species A RD” PCR tube. Place both tubes on ice.
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- Using a micropipette and a fresh tip, pipette 10 µl of PCR product from the “Species B” PCR
tube into the “Species B RD” PCR tube. Place both tubes on ice.
- Using a micropipette and a fresh tip, pipette 10 µl of PCR product from the “White” PCR
tube into the “White RD” PCR tube. Place both tubes on ice.
- Place your “Species A RD”, “Species B RD”, and “White RD” tubes into the 37C water
bath for 45-60 minutes. Remove tubes from the water bath and place on ice.
► What is the function of the water bath? (Keep in mind that HaeIII is an enzyme)
3.
Setting up Gel Electrophoresis
- Using a micropipette and fresh tip, pipette 4 µl of loading dye into the “Species A RD” tube.
Flick the tube to ensure that the loading dye is mixed evenly into solution.
- Using the same pipette tip, pipette 30 µl of the solution in the “Species A RD” tube into one
of the wells in the gel. In the space below, record the well number you loaded your sample
into (always read from left to right).
Species A Well Number: _________
- Using a micropipette and fresh tip, pipette 4 µl of loading dye into the “Species B RD” tube.
Flick the tube to ensure that the loading dye is mixed evenly into solution.
- Using the same pipette tip, pipette 30 µl of the solution in the “Species B RD” tube into one
of the wells in the gel. In the space below, record the well number you loaded your sample
into.
Species B Well Number: _________
- Using a micropipette and fresh tip, pipette 4 µl of loading dye into the “White RD” tube.
Flick the tube to ensure that the loading dye is mixed evenly into solution.
- Using the same pipette tip, pipette 30 µl of the solution in the “White RD” tube into one of
the wells in the gel. In the space below, record the well number you loaded your sample into.
White Button Well Number: _________
- After all of the wells have been loaded, run the gel at 90V for 30-45 minutes.
- Use gloves to carefully remove the gel from the gel box and place in the UV light box.
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Sketch your observations in the space below.
Load here
Molecular weight markers
Species A
Species B
White button mushroom
► Compare your results to the sketch of expected results that you produced earlier. Based
on the banding patterns you observed on the gel, what is the identity of Species A?
Species B?
► Did the results for the button mushroom match what you expected? Explain.
Acknowledgments:
This laboratory exercise was modified from the following sources by Katie D’Amico, EFB MS
candidate, March 2010, and Yazmin Rivera, EFB PhD candidate, March 2011.
Martin, P., et al. 2004. A Rapid PCR-RFLP Method for Monitoring Genetic Variation Among
Commercial Mushroom Species. Biochemistry and Molecular Biology Education, 32(6), 390-94.
New England Biolabs, Inc. (2010). [Gel illustration of the 100 bp molecular weight marker.] 100
bp DNA Ladder(N3231), DNA Markers/Ladders, NEB. Retrieved from
http://www.neb.com/nebecomm/products/productN3231.asp.
Raven, P.H., et al. 2008. Biology (8th ed.). McGraw-Hill. New York, New York.
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