Restriction Enzyme Analysis of DNA
Austin CaoMr. Savage, AP BiologyS5S4S3S2S1CS
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-31) What
is the sequence of the complementary DNA strand?
3-TTTCAGCGACCTTAAGTGACGTAGCTTAAGGGCCCCGATATATACCTTAAGCT-52) 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.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 Digest1) 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 Electrophoresis1) 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 DNAElectrophoresis Data
Lambda/HindIII size standardCrime SceneSuspect 1Suspect 2Suspect
3Suspect 4Suspect 5
BandDist.(mm)size (bp)Dist.(mm)size (bp)Dist.(mm)size
(bp)Distmm)size (bp)Dist.(mm)size (bp)Dist.(mm)size
(bp)Dist.(mm)size (bp)
12023,130353287392922392922353287392922392922
2249,416392922502681462717382980522673442732
3286,557562637532657522673552637622607522673
4344,361622607
5422,322
6442,027
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 Results1) 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 ResultsHINDIIIBAMHIECORI
DistanceBP LengthDistanceBP LengthDistanceBP Length
2.6*27,4912.82.7
2.6*23,1303.13.8
3.49,4163.84.2
4.06,5574.04.5
4.74,3614.35.3
6.52,322
7.02,027
Section 8: Designing your own experimentI 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 7th 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.
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 resultsThe 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
BiologyScience 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 Mapping1) From the map of plasmid S2 list
all the restriction enzymes that would cut this plasmid. PvuLL,
EcoRI, BamHI, PstI, ScaI, HindIII2) Which plasmid S2 or S5, is the
biggest and what is its size? S5 is 9481 bp3) 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, 26145) 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 I8) 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 PlasmidYou 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? 94812) 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 S54) 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) I7)
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, 8638) 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) I9) 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 Plasmid1) Where is the Pstl site on
the pTZ18U plasmid? 2982) Look at plasmid S4. What segment of the
lambda bacteriophage has been inserted? 5218-96173) 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? 34) 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, yes5) Where is the EcoRl site on the
parent pTZ18U plasmid? 2556) Choose a segment of lambda
bacteriophage genome that could be cut out by the EcoRl enzyme.
Which segment will you use? 21226-261047) How big is your new
plasmid? 77388) 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: 2a) EcoRl alone 2860, 4878b)
Pstl alone 3722, 4016c) EcoRl and Pstl together 2817, 3722, 11999)
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.