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lambda DNA Fingerprinting Simulation

By Thomas J. Conley



Type of Entry:

  • Hands-on
  • Simulation

Type of activity:

  • Hands-on
  • Simulation

Target audience:

  • Biology (Biotechnology, Advanced Biology)

Preparation time:

2-3 hrs if teacher prepares all of the agarose gel beds. Make gels first and while they are solidifying aliquot out your DNA, restriction enzymes, 2X restriction buffer and loading dye. Remember to keep DNA and enzymes on ice. Return to freezer until ready to use.


Abstract

The purpose of this lab activity is to demonstrate (through simulation) how DNA fingerprinting (or DNA profiling) might be used to solve a crime. In this activity, students perform restriction digests on DNA samples from four individuals, and then search for similarities between the individuals by running the restriction fragments on an electrophoresis gel. Because not two people ( except identical twins) have exactly the same DNA, a person's DNA fingerprint is unique and can be used for purposes of ide ntification. This activity does not do a true DNA fingerprint. It simulates two of the three steps of DNA fingerprinting: restriction of DNA sample and separation by electrophoresis. This activity does not make use of the third step, the radioactive probes. In ord er to make DNA fingerprinting affordable, lambda DNA is used instead of plasmids. This means that the teacher has to switch the labels on the samples given to the students. What is labelled DNA is actually the different restriction enzymes and what is l abelled restriction enzyme is the lambda DNA. Although there is some deception on the part of the teacher, the students are able to do a restriction digest that simulates a crime scene which adds interest for the student.


Background

This lesson was developed with my Woodrow Wilson Bio-4 TORCH team which included Bill Bandura, Karen Kyker and Myrtle Brijbasi


Project

Notes for teacher:

For the purposes of this activity, here is the scenario: A murder has been committed, and police discover evidence of a struggle and blood traces at the scene of the crime. Suspects X, Y, and Z are arrested and will go through DNA profiling tests to determine if they were at the scene of the crime. All of the suspects proclaim their innocence adamantly, and want to see their lawyers. At their indictments, it is learned that

  1. suspect X, a star athlete and former husband of the deceased, claims to have been out of town when the crime was committed

  2. suspect Y, current boyfriend of the deceased, said he was home alone at the time of the murder

  3. suspect Z, a pizza delivery boy, says that he was out delivering pizzas in a neighborhood other than where the murder took place.

At the start of this exercise, the instructor furnishes the students with samples of DNA from the three individuals involved and DNA isolated from blood samples collected at the crime scene. Remember that what you are really giving them is the different restriction enzymes. The students then follow the protocol for a restriction digest of these samples using the restriction enzyme (which is the lambda DNA) provided and loading them on an electrophoresis gel. After electrophoresing the samples and then s taining the gels to see the DNA bands, students may then determine which of the suspects is the likely murderer based on forensic evidence.

Class time needed:

  • 55 minute period to digest and load DNA. It helps to give them aliquoted samples of what we are calling the DNA in four labelled tubes.

  • 1 hour to run gels which would not have to be done in class. Teacher can remove gels after they have run sufficiently and place them in baggies or saran wrap until ready to do the staining.

  • 55 minute period to stain and destain the gels, and to analyze. Teacher can shorten this time period by doing the staining and destaining if not able to devote to full periods to this activity.

Required of students:

Students need to have some prior knowledge of recombinant DNA technology and DNA fingerprinting. If students have not used a micropipettor and have not loaded samples into a gel, then time must be spent practicing these techniques prior to doing this lab . To practice loading wells the teacher can pour some agarose into petri dishes that have combs in place. Students can use a mixture of loading dye and water to practice loading gels.


lambda DNA Fingerprinting Simulation

Teacher Information

Materials: (for 30 students, divided into 10 groups of 3 students)

  • 40 ul. EcoRI
  • 20 ul. BamHI
  • 20 ul. HindIII
  • 120 ul. lambda DNA (0.5 mg/ml)
  • 200 ul. 2x compromise restriction buffer
  • 20 ul. loading dye
  • 0.8% agarose (4 g. agarose/ 500 ml. TBE buffer)
  • 5 liters 1X TBE Buffer
  • Carolina Blue stain
  • 50 microcentrifuge tubes
  • 10 0.5 - 10 ul. micropipettors and tips
  • 37oC water bath
  • 10 sets electrophoresis equipment
  • 10 microtube racks
  • hot plate with magnetic stirrer or microwave oven

Teacher Preparation:

KEEP IN MIND THAT THE DNA SAMPLES IN THIS LAB ARE ACTUALLY DIFFERENT RESTRICTION ENZYMES AND THE RESTRICTION ENZYME IS ACTUALLY LAMBDA DNA!!

  1. DNA sample tubes for suspects X, Y, and Z as well as from blood samples collected at the scene of the crime (E) should be prepared as follows:

    suspect  X DNA:     20 ul. HindIII      (X)
    suspect  Y DNA:     20 ul. EcoRI        (Y)
    suspect  Z DNA:     20 ul. BamHI        (Z)
    evidence DNA:       20 ul. EcoRI        (E)

    (2 ul. aliquots in 1.5 ml tubes may be supplied to students to save time and materials)

  2. The 120 ul. of l DNA (0.5mg. / ul. ) should be aliquoted 12 ul. per lab group and be labeled "restriction enzyme" (R).

  3. The 200 ul. of compromise restriction buffer should be aliquoted 20 ul. per lab group and labeled "buffer" (B).

    We used the BamHI buffer as our compromise buffer and had good results.

  4. 0.8% agarose may be prepared by combining 4 grams of agarose with 500 ml. TBE buffer, microwave, and stir until suspended. Heat and keep at 60oC using water bath. Allowing 40-50 ml. per gel, this should be enough to pour at least 10 gels.

Analysis:

DNA profiling, or as it is more commonly called, DNA fingerprinting, involves three basic steps-- restriction of the DNA samples into fragments that can be handled more easily: separation by size of the various length fragments using electrophoresis; and then visualizing certain fragments to which radioactive probes have been attached.

A review of DNA fingerprinting in general would be appropriate at this time. Keep in mind that this lab only simulates an actual DNA fingerprint, and so the final step using probes will not be done.

Compare the DNA profiles of the suspects in this case to the DNA profile of the blood found at the crime scene. Which suspect does this test put at the crime scene?


Student Lab Sheet
Name:

Introduction:

A murder has been committed, and police discover evidence of a struggle and blood traces at the scene of the crime. Suspects X, Y, and Z are arrested and will go through DNA profiling tests to determine if they were at the scene of the crime. All of th e suspects proclaim their innocence adamantly, and want to see their lawyers. At their indictments, it is learned that

  1. suspect X, a star athlete and former husband of the deceased, claims to have been out of town when the crime was committed

  2. suspect Y, current boyfriend of the deceased, said he was home alone at the time of the murder

  3. suspect Z, a pizza delivery boy, says that he was out delivering pizzas in a neighborhood other than where the murder took place.

    You are the lab worker who has been handed the DNA samples from the three suspects involved plus the DNA from the blood at the crime scene. Using molecular biology techniques, your job is to determine which of the suspects might have been at the crime sc ene. The court awaits your decision.

    Materials:

    DNA samples ( 2 ul. each of four samples)

    • 12 ul. restriction enzyme
    • 20 ul. 2X restriction buffer
    • 4 ul. loading dye
    • 500 ml. TBE buffer
    • 50 ml. 0.8% agarose for gel
    • 0.5 - 10 ul. micropipettor and tips
    • Carolina Blue stain
    • 37oC water bath
    • microcentrifuge tube rack
    • electrophoresis equipment (power supply, gel box, 8 tooth comb)

    Procedure:

    1. Obtain from your instructor the 2 ul. samples of DNA from each of the suspects (X, Y, and Z) and a 2 ul. sample from the blood droplets at the scene of the crime (E).

    2. Using a fresh tip, add 5 ul. 2X restriction buffer (B) to each tube.

    3. Using a fresh tip, add 2 ul. restriction enzyme (R) to each tube. Close tops of tubes and spin down by pulsing in a microcentrifuge to mix. If you have no microcentrifuge, tap sharply on table to mix droplets.

    4. Place in a 37oC water bath and incubate for a minimum of 30 minutes. After incubation, digests may be frozen and saved for another day.

    5. Dam ends of casting tray with masking tape. Place comb in place.

    6. Carefully pour enough agarose to make a gel 5 mm. thick (about 1/3 the way up comb teeth). After pushing bubbles out of the way with a pipettor tip, put casting tray out of the way to set undisturbed for 10-15 minutes.

    7. After gel has set, remove tape from ends of casting tray. Put casting tray in gel box so that comb end is closest to negative (black) electrode.

    8. Pour enough TBE buffer into gel box so that gel is just covered.

    9. Gently remove comb, being careful not to tear gel.

    10. Make sure wells are completely submerged; add more buffer if needed. If gel box is covered, gel may be left for several days.

    11. Add 1 ul. loading dye to each tube and close tops. Mix by pulsing in microcentrifuge, drawing in and out with a micropipettor, or tapping on table.

    12. Load entire contents of each sample tube into separate well in gel. Be sure micro-pipettor tip is below surface of buffer and just above the center of each well that you load.

      Leaving two empty lanes on both sides, load in the order shown below:

           X	         Y          Z          E

      X = former husband

      Y = boyfriend

      Z = delivery boy

      E = evidence DNA

    13. Once the wells are loaded, put the top on the gel box and connect it to the power supply. Plug in the power supply and turn the unit on. Adjust to 100-150 volts and run for 30-40 minutes (loading dye should almost completely cross gel). At this point the current may be shut off and the gel stained to visualize the DNA bands.

    14. Follow instructions for staining gel with Carolina Blue.

    Analysis:

    Compare the fingerprints of all the suspects in this case to the DNA profile of the DNA isolated from the blood droplets at the crime scene. Which suspectıs blood was found at the site of the murder?

    Draw a sketch below of your gel with the fingerprints on them.


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