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Restriction Enzyme Analysis of DNA

Austin Cao
Mr. Savage, AP Biology

Section 1: Restriction Enzymes
Restriction enzymes cut DNA strands at specific sequences that are recognized. Ultimately, using these
enzymes, we can cut up a strand of DNA into a collection of various-sized pieces. Running the fragments
through a gel electrophoresis will separate our DNA pieces by length. Thus the specific composition of the
original DNA strand is revealed. This can be used to identify DNA when related questions are brought up:
whose DNA is in the coffee cup left at the crime scene? Who is the father? What animal meat is in your
sandwich?

5-AAAGTCGCTGGAATTCACTGCATCGAATTCCCGGGGCTATATATGGAATTCGA-3
1) What is the sequence of the complementary DNA strand?
3-TTTCAGCGACCTTAAGTGACGTAGCTTAAGGGCCCCGATATATACCTTAAGCT-5
2) The restriction site for EcoRI is 5-GAATTC-3 and the enzyme makes a staggered cut between G
and A on both strands. Draw an illustration showing how the DNA fragment is cut by EcoRI.
5-AAAGTCGCTGGAATTCACTGCATCG(cut)AATTCCCGGGGCTATATATGGAATTCGA-3
3-TTTCAGCGACCTTAAGTGACGTAGCTTAA(cut)GGGCCCCGATATATACCTTAAGCT-5

Section 2:DNA Mapping:
1) Can you make a prediction about the products of DNA from different sources cut with the same
restriction enzymes?
While 99% is usually the same sequences, there will be differences between samples. So the
same restriction enzymes could create different RFLP patterns for different sources.
2) Will the RFLP patterns produced by electrophoresis produced by DNA mapping be the same or
different if you use just one restriction enzyme?
The probability that the single enzyme will cut at the piece of DNA that differs between
sources is highly unlikely.
S5
S4
S3
S2
S1
CS
3) Do you have to use many restriction enzymes to find differences between individuals? Justify your
prediction.
Yes, different enzymes make the RFLP more accurate, revealing differences in sequences.
4) Can you make a prediction about the RFLP patterns of identical twins cut with the same restriction
enzymes?
Identical DNA implies identical RFLPs.
5) How about the FRLP patterns of fraternal twins or triplets?
Non-identical DNA implies non-identical RFLPs.

Section 3: Restriction Digest
1) Before you incubated your samples, describe any visible signs of change in the contents of the tubes
containing the DNA after it was combined with the restriction enzymes.
No visible change.
2) Can you see any evidence to indicate that your samples of DNA were fragmented or altered in any
way by the addition of Eco-RI/Pstl? Explain.
No, DNA is extremely small and fragmentation would be impossible to see.
3) In the absence of any visible evidence of change, is it still possible that the DNA samples were
fragmented? Explain your reasoning.
Yes, the fragmented pieces wont separate neatly by size until we run the electrophoresis.
4) After a 24 hour incubation period, are there any visible clues that the restriction enzymes may have
in some ways changed the DNA in any of the tubes? Explain your reasons.
No, I cannot see the DNA or any clues that it was changed.

Section 4: Agarose Gel Electrophoresis
1) To which electrode would you expect DNA to migrate? Explain.
Because DNA is negatively charged, it is attracted to the positive anode.
2) What color represents the negative pole?
Black.
3) What size fragments would you expect to move toward the opposite end of the gel most quickly?
With less resistance, smaller fragments can travel through the agarose matrix quicker.
4) Which fragments are expected to travel the shortest distance from the well. Explain.
Larger fragments find it harder to move. They are physically slowed down by the matrix.

1) What can you assume is contained within each band?
DNA fragments.
2) If this were a fingerprinting gel, how many samples of DNA can you assume were placed in each
separate well?
Most likely a couple samples from the same source to ensure solid results.
3) What would be a logical explanation as to why there is more than one band of DNA for each of the
samples?
Fragments of similar lengths group together form bands.
4) What caused the DNA to become fragmented?
The restriction enzyme.
5) Which of the DNA samples have the same number of restriction sites for the restriction
endonucleases used? Write the lane numbers.
Lanes 1, 2, 3, 4, 5 all had three bands.
6) Which sample has the smallest DNA fragment?
Lanes 5, 6 had the smallest DNA fragment.
7) Assuming a circular piece of DNA was used as starting material, how many restriction sites were
there in lane three?
Two.
8) From the gel drawing on page 35, which DNA samples appear to have been cut into the same
number and size of fragments?
Lanes 2, 4.
9) Based on your analysis of the sample gel drawing, what is your conclusion about the DNA samples
in the drawing? Do any of the samples seem to be from the same source? If so, which ones?
Describe the evidence that supports your conclusions.
Suspect 2 matches the sample from the crime scene. Because their RFLPs are identical, we
reason that they are from the same source of DNA.

Section 5: Quantitative analysis of DNA
Electrophoresis Data
Lambda/HindIII
size standard
Crime Scene Suspect 1 Suspect 2 Suspect 3 Suspect 4 Suspect 5
Band Dist.
(mm)
size
(bp)
Dist.
(mm)
size
(bp)
Dist.
(mm)
size
(bp)
Dist
mm)
size
(bp)
Dist.
(mm)
size
(bp)
Dist.
(mm)
size
(bp)
Dist.
(mm)
size
(bp)
1 20 23,130 35 3287 39 2922 39 2922 35 3287 39 2922 39 2922
2 24 9,416 39 2922 50 2681 46 2717 38 2980 52 2673 44 2732
3 28 6,557 56 2637 53 2657 52 2673 55 2637 62 2607 52 2673
4 34 4,361 62 2607
5 42 2,322
6 44 2,027

Then underneath the data table insert both the semi log and regular graph with the scale you had to create
for your gel. (You will need your own distance migrated scale entered. These graphs have a text box embedded so
you can change the numbers to match your gel.)

Section 6: Interpretation of Results
1) What are we trying to determine? Restate the central question.
The central question is: whose DNA matches the DNA collected at the crime scene?
2) Which of your DNA samples were fragmented? What would your gel look like if the DNA were not
fragmented?
All of our samples were fragmented. Each lane would have a single band.
3) What caused the DNA to become fragmented?
The restriction enzymes.
4) What determines where a restriction endonuclease will cut a DNA molecule?
The enzymes look for a certain nucleotide sequence. Its determined by chemical triggers.
5) A restriction endonuclease cuts two DNA molecules at the same location. What can you assume
is identical about the molecules at that location?
The have the same nucleotide sequence on either side of the cut.
6) Do any of your suspect samples appear to have EcoRI or Pstl recognition sites at the same location
as the DNA from the crime scene?
Yes, suspect 3.
7) Based on the above analysis do any of the suspect samples of DNA seem to be from the same
individual as the DNA from the crime scene? Describe the scientific evidence that supports your
conclusion.
The RFLP from suspect 3 matches the RFLP from the crime scene. Same number of bands,
same thickness of each. We conclude that the original DNA was thus identical.

Section 7: Analyzing Results
HINDIII BAMHI ECORI
Distance BP Length Distance BP Length Distance BP Length
2.6 *27,491 2.8 2.7
2.6 *23,130 3.1 3.8
3.4 9,416 3.8 4.2
4.0 6,557 4.0 4.5
4.7 4,361 4.3 5.3
6.5 2,322
7.0 2,027


Section 8: Designing your own experiment
I would collect the three DNA samples: Ms. Masons coffee cup, Mr. Gladsons tissue, and Bobbys gum.
Along with the HindIII ideal sample, I would mix them with the restriction enzymes, incubate, load the
dye, and then run them in the electrophoresis. I would identify the matching RFLPs to match the DNA,
thus finding the owner of the spilt blood. The DNA would match Mr. Gladson. Circumstantial evidence,
like the rusty stains on his lab coat and the Erlenmeyer flask, would also point to Mr. Gladson. The email
would support this conclusion as well.

But when we traced the IP address of the email, it came up in a small neighborhood outside of Boston:
Bobbys house. When we got to his house, we found a body: it was Laurel. Bruises on her body and DNA
evidence indicated that Bobby had raped and murdered her. I was horrified, but I persisted. The mystery
was too enticing. Too many lives were at stake. And I knew I could save them. In Bobbys room we found a
vial of blood. It was Mr. Gladsons. Mr. Gladson told us that last week, he had gotten a paper cut. Bobby
had eagerly helped him wipe off the blood. Ah, so Mr. Gladson had been framed!

We knew Bobby had an addiction to Bubble gum, so we closed off the area and searched nearby gas
stations. Sure enough, one of our officers spotted him at a 7-11, drinking one of their 59 cent medium
Slurpies. A car chase ensued, but we lost him when he ran into the subway station.

Suddenly, our office got a call. There had been an explosion off of 7
th
and Main. It came from underground.
From the subway tunnels. Initial reports indicated 40 dead, and over 100 missing. In the aftermath we
made two critical discoveries: 1) there were traces of dormant radioactive material (not enough to harm
anyone), and 2) there were traces of DNA from the Chukar bird. The Chukar bird was native to Pakistan.
Our worst fears were coming true: Bobby, the talkative white boy we all had loved, was in a U.S-based
sleeper cell for Al Qaeda.

1
10
100
1,000
10,000
100,000
0 1 2 3 4 5 6 7 8
B
P

L
e
n
g
t
h

Distance
electrophoresis to base pair length
Reports started coming in concerning the JFK International Airport: communications had been severed,
and a hostage situation seemed to be evolving. Calls came flooding in, as panicked travelers found
themselves trapped in the closed off airport. But it didnt seem as if this was a group of thugs with guns
because no one could get out. Something else must have been happening in that airport. As I stood outside
the main doors, unable to see inside, I knew that we would need to take action and fast.

Before I could do anything however, a new situation began developing overseas. One of our agents
assigned in Russia had been taken out. His job had been to assist the Russians in protecting one of their
older nuclear warheads turns out we should never have trusted them. Putin called: they had killed our
agent in order to reactivate the nuke. Damn him! In their efforts to add more nukes to their arsenal, they
had exposed themselves to Chechen terrorists who took the opportunity to steal the newly activated nuke.
And now it was sitting inside JFK, in the middle of New York. The Chechens now controlled Americas Big
Apple. Like many of my fellow Americans, I knew that I would do anything to save my apples.

I was angry. I was mad. Infuriated. Enraged. Fuming. Riled. As I drove back to the airport, I thought of
synonyms for how I felt. My therapist told me to repeat these words to calm myself down. But I was livid.
So I stormed in to the airport with a Glock and single handedly took out all of the terrorists. And there in
the middle of the airport stood Bobby, one hand was on the trigger to the warhead, the other was stroking
the beard he had grown in less than 2 hours. I didnt even recognize him anymore. He yelled something in
Arabic that he probably had picked up from Google Translate, and tried to press the trigger. But I shot his
hand off. The mission was over. Bobby was arrested. We had saved America.
My name is Jack Bauer, and this has been the longest day of my life.

Section 9: Thinking about your results
The Innocence Project tries to exonerate prosecuted people through DNA testing. Through its 20 years of
existence, 311 people have already been freed of wrong convictions. Its funded by donations. Its philosophy
is rooted in a belief that incorrectly accusing someone of a crime is the greatest crime of all (I made up that
lineits pretty good).

One United States Supreme Court justice expressed concern that DNA testing poses risks to the criminal
justice system. That was Chief Justice Roberts. What he fails to understand is that if our criminal justice
system does in fact incarcerate innocent people, then it is not judicial. So yes, DNA testing will upheave
the status quo. And that is good. Let it overthrow the current system and replace it with a better one.
So ultimately there is only a single ethical issue raised by DNA exoneration cases: should the criminal
justice system do everything it takes to find the truth and ensure justice, or should it do everything it takes
to ensure that it doesnt change?

Chief Justice Roberts is a great guy. He also has some foolish opinions. Lets walk through them. It will be
fun. On the D.C. Circuit he dealt with a case in which a 12-year-old girl was arrested, searched, and
detained for eating a single fry in a subway station that had a no eating rule. Her mom sued, saying that
the arrest infringed on the 4th and 5th Amendments. He dismissed the case. In Gonzales v. Oregon,
Roberts opposed physician assisted suicide for terminally ill patients. In Georgia v. Randolph, he argued
that police can search a house without a warrant, when one occupant consents and the other does not. In
Morse v. Fredrick, he ruled that schools could suppress student speech that did not agree with the school.
However, in U.S. v. Stevens, he also ruled that videos depicting animal murder and torture were protected
by the First Amendment, and could be legally sold.

Hes said before that "We are not asked to say whether we think this law is unwise, or even asinine we
are asked to hold that it violates the United States Constitution. He is unable to reform the very system
that he works in, or see the flaws inherent in its operation. Although DNA testing will clearly result in
more accurate prosecutions and a better system, it will also delegitimize established traditions in the
criminal system, like the decision factor of eyewitness testimony. That fundamental questioning of the
system, he will not allow.

The two questions that the packet asks, what social issues are raised by using DNA evidence, and what
other arguments can you make against using DNA evidence, are rooted in this same paradigm of resisting
change. So stick it to the man.

Section 10: Where can you go from here?
Science from AP Biology Science from Rectify
Mr. Savage gave us samples of DNA. DNA was swabbed from the dead girls body.
We were able to match the crime scene RFLP with
that of a suspects, Ryan Brink.
The testing could only conclude that it was not
Daniel Holden.
We did it in a couple days. They took 20 years to finally find the DNA evidence.
We trusted that the DNA evidence was proof of
guilt.
In the show, DNA evidence is not enough to
suppress the case forever.
All we cared about was finding the perpetrator. Whether Daniel did the crime or not is irrelevant to
the show.
This is a trailer for Rectify: http://www.imdb.com/video/imdb/vi3314329113/

Section 11: Plasmid Mapping
1) From the map of plasmid S2 list all the restriction enzymes that
would cut this plasmid.
PvuLL, EcoRI, BamHI, PstI, ScaI, HindIII
2) Which plasmid S2 or S5, is the biggest and what is its size?
S5 is 9481 bp
3) Using plasmid S2 as an example, find the restriction sites for the
enzyme Pvull. How many sites are there? What is their location?
If Pvull was used to cut this plasmid how many fragments would
it make?
There are 3 cuts, so 3 fragments.
4) Next determine the size of the fragments created when plasmid S2
is cut by Pvull. DNA fragment size is calculated by subtracting the site locations from each other.
(Note: if a fragment contains the 0 point of the plasmid, it is not just a simple subtraction!). How
big are the fragments from plasmid S2 that is cut with Pvull? The fragment sizes should add up to
the total for that plasmid (5869 bp).
1938, 1417, 2614
5) If the fragments from the plasmid S2 digested with Pvull were run on an agarose gel, what would
they look like? Draw the gel and label the fragments and their sizes.
There would be three bands. The ones farther away would be smallest.
6) Now you can determine the fragment sizes of the plasmids when cut with the two enzymes, EcoRi
and Pstl. Indicate the sizes of the fragments that would be generated if the plasmid were a digest by
Pstl alone, EcoRI alone of by both Pstl and EcoRI. This is a table:
Pstl: 1700, 150, 1159, 3158 (4)
EcoRI: 5869 (1)
Both: 43, 1700, 150, 1159, 3072 (5)
7) If plasmid S2 was digested and run on an agarose gel, what would the gel look like? Draw a gel and
the fragment sizes if digested by EcoRI alone, Pstl alone and by EcoRI and Pstl together.
Pstl: I I I I
EcoRI: I
Both: I I I I I
8) How does your diagram in question 7 compare to what was observed in your gel after the
experiment Indicate a reason for why your data in question 7 might be different from the actual
experimental data seen from lesson 2.
Distance to bp is logarithmic I cant plot by hand a log scale.

Section 12: Mapping the Plasmid
You will need to leave room under question 6 to draw your PstI fragment.
1) How big is plasmid S5? Add the fragments in each column. The total
should add up to the size of the plasmid. Why?
9481
2) How many fragments are there? Did the enzyme cut the plasmid, or
did it remain as a circle? How could you tell?
However many cuts there are, there are fragments. Yes, the
plasmid was cut.
3) Compare the data from the Pstl digest of plasmid S5 with that of the
EcoRI digest. How many fragments are there? How many restriction sites are there for Pstl?
7 in S5
4) How many fragments are there when EcoRI and Pstl are used to digest plasmid S5? Does that
answer the question of whether or not EcoRI cut the plasmid? Why?
8, yes it did. There is a cut shown.
5) Which fragment of Pstl digested plasmid S5 was shortened by a cut with EcoRI? How do you know
this?
From 6919 to 298. It is shown on the chart.
6) Draw the Pstl fragment that is cut with EcoRI in plasmid S5 to demonstrate how the fragment was
cut with EcoRI.
I I(cut) I
7) Shown above is the data generated from digestion of plasmid S3 with EcoRI and Pstl. How many
times did EcoRI cut plasmid S3? What are the fragment sizes?
2, 6504, 863
8) The data from the EcoRI digest of plasmid S3 indicate that the fragments are not equal. Draw a
possible map and label the EcoRI sites and the sizes of the fragments.
I (863) I (6504) I
9) Now draw an approximate map of the Pstl sites on plasmid S3 and label the Pstl sites and the sizes
of the fragments.
I (2860) I (4507) I
10) Is there another possible order of restriction sites on plasmid S3 digested with both Pstl and EcoRI?
How might you resolve these possibilities?
Yes, use another restriction enzyme to check.
11) When the gels were run for this experiment, there were only three bands for plasmid S3. Which
band is missing from your gel? Why?
43 is really small. So its missing.

Section 13 Constructing a Plasmid
1) Where is the Pstl site on the pTZ18U plasmid?
298
2) Look at plasmid S4. What segment of the lambda bacteriophage has been inserted?
5218-9617
3) After looking at the plasmid map and also the lambda phage map, can you determine how many Pstl
restriction sites were added to the plasmid because of the inserted lambda phage DNA fragment?
3
4) Look at plasmid S1. What segment of lambda was added to that plasmid? Were any Pstl restriction
sites added to the plasmid with the inserted fragment of lambda DNA?
20285-22425, yes
5) Where is the EcoRl site on the parent pTZ18U plasmid?
255
6) Choose a segment of lambda bacteriophage genome that could be cut out by the EcoRl enzyme.
Which segment will you use?
21226-26104
7) How big is your new plasmid?
7738
8) How mamny restriction sites are there now for PStl in your new plasmid? Predict what fragments
you would generate if you were to digest your plasmid with:
2
a) EcoRl alone
2860, 4878
b) Pstl alone
3722, 4016
c) EcoRl and Pstl together
2817, 3722, 1199
9) Draw and agarose gel for each of these digests and label the fragment sizes.
EcoRI: I(2860) I(4878)
Pstl: I(3722) I(4016)
Both: I(1199) I(2817) I(3722)
10) How could you use plasmid mapping to determine in which orientation your fragment was inserted?
Compare them to the lambda fragments.

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