Restriction Enzyme Analysis of DNA

Austin CaoMr. 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:

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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 won’t 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 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)

Distmm)

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 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. It’s 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 ResultsHINDIII BAMHI ECORI

Distance BP Length Distance BP Length Distance BP Length2.6 *27,491 2.8 2.72.6 *23,130 3.1 3.83.4 9,416 3.8 4.24.0 6,557 4.0 4.54.7 4,361 4.3 5.36.5 2,3227.0 2,027

2 3 4 5 6 7 81,000

10,000

100,000

electrophoresis to base pair length

Distance

BP

Len

gth

Section 8: Designing your own experimentI would collect the three DNA samples: Ms. Mason’s coffee cup, Mr. Gladson’s tissue, and Bobby’s 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: Bobby’s 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 Bobby’s room we found a vial of

blood. It was Mr. Gladson’s. 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 didn’t 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 America’s 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 didn’t 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. It’s 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 line…its 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 doesn’t change?

Chief Justice Roberts is a great guy. He also has some foolish opinions. Let’s 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.

He’s 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 RectifyMr. Savage gave us samples of DNA. DNA was swabbed from the dead girl’s

body.We were able to match the crime scene RFLP with that of a suspect’s, 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, HindIII

2) 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 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 can’t 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) 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, 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) 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 it’s 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? 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, 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? 21226-26104

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:

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.