Materials Included In Kit

Additional Materials Required

Safety Precautions

Pop beads and their connectors are small and fragile. Do not allow any ingestion of materials.

Disposal

All materials are reusable many times.

Teacher Tips

• This laboratory should not be attempted until the basic structure and functioning of DNA has been covered.

• This lab activity can be completed in one class period if teams are organized and well prepared. All materials are reusable. It might be logical to organize and discuss the lab one day and conduct the lab on a second day.
• Time can be saved by counting and distributing pop beads prior to class time. Use cups to hold the various colors of pop beads.
• If students reverse their DNA pop bead strand during their work they will not produce logical or correct autoradiograms. Emphasize the need to keep 3'–5' orientations. Refer students back to the DNA Sequence Worksheet if they mix up their DNA strands.
• The physical manipulations of the pop bead DNA molecules is critical and helpful in making DNA fingerprinting procedures more concrete. Do not allow students to complete the exercise on paper just to get the right answer.
• Make copies of each of the DNA Sequence Worksheet, Paper Gel Electrophoresis Template and Autoradiogram Worksheet for each student.

Answers to Questions

4. Compare all the DNA fingerprints. Can the guilty suspect be identified from the DNA fingerprints? Answer the following questions on the back of the Autoradiogram worksheet.

1. Based on the autoradiogram’s results, which suspect is the murderer?
   

   Suspect 1 is the murderer. Suspect 1’s DNA banding pattern is identical to that of the DNA collected at the crime scene.
   

2. Discuss with your investigative team the potentially controversial aspects of DNA fingerprinting. Write a summary of the key discussion points.
   

   In court cases, many issues may be somewhat unrelated to the actual scientific evidence. The handling of the evidence by police officers and crime lab employees is often challenged and disputed. [Chain of Custody] The actual procedures of producing the DNA fingerprints can be difficult for jurors to understand and the confusion can lead to a disregard for the scientific evidence. Questions about the calculated odds of two individuals having the same banding patterns can be difficult to justify “beyond a reasonable doubt.” What is reasonable doubt? [One in a million, a billion, a trillion?]
   

3. How might DNA fingerprinting be used to determine if a child belongs to a specific parent? What might a child’s autoradiogram look like compared to a mother and father’s?
   

   DNA fingerprinting can prove familial relationships with a high degree of certainty. If DNA fingerprints can be secured for a child and questionable parents, the connections can be easily established since a child gets half of its genome from its mother and the other half from it father. All bands in the child’s fingerprint can be accounted for in the child’s real parents.

Teacher Handouts

10332_Teacher1.pdf

Introduction

Today, DNA fingerprinting is used routinely to test samples of blood, hair, saliva and other bodily fluids left at crime scenes. What is DNA and how can it be “fingerprinted”?

Concepts

• DNA structure

• Restriction Fragment Length Polymorphism (RFLPs)
• Polymerase Chain Reaction (PCR)
• Autoradiogram
• DNA fingerprint

Background

Less than 50 years ago the nature of the genetic code still eluded scientists. In the 50 years since the structure of DNA was first unraveled, it has become the most significant biological topic of the century. Understanding the structure of DNA helps to explain many life processes and leads to greater knowledge of why we are who we are. In addition, the uniqueness of every individual organism’s DNA can be used as a tool to discover relationships between organisms. This activity looks at how DNA analysis can be used to uncover criminal relationships.

A simplified diagram of a short section of DNA is shown in Figure 1. The diagrammed segment contains seven base pairs. A real chromosome may contain a single DNA molecule with as many as 10⁸ (100 million) base pairs or even more! Since the base pairs represent the genetic code, the chromosomes can store a lot of genetic messages!

This activity uses short simulated DNA segments—remember real DNA analysis involves millions of base pairs.

Understanding the process of DNA fingerprinting requires knowledge of DNA beyond its basic coding structure. It requires knowing further properties of the DNA found in chromosomes. The key to understanding how each person can have totally unique DNA resulted with the discovery of introns. Introns are repeated sequences of “nonsense” or “junk” DNA found all along the length of DNA molecules. Introns do not code for a specific protein like the key “genes” in the key coded sections. Individuals have very similar gene sequences in their DNA but have tremendous variation in the length and number of introns. Introns are made up of repeating DNA base pair sequences and the number of repetitions varies from person to person. These repetitive intron fragments are called “Restriction Fragment Length Polymorphisms” (RFLPs). RFLPs are unique to each person, except in the case of identical twins.

DNA fingerprinting procedure is conducted using the following key steps are involved and simulated in this activity.

1. DNA is first extracted, typically from tissue samples such as blood, skin, hair, semen or saliva.
2. If insufficient DNA is found in the sample it can be amplified (multiplied) by a technique called “Polymerase Chain Reaction” (PCR). This procedure capitalizes on DNA’s natural duplicating capability. DNA from only a few cells can be amplified to generate enough material for fingerprint analysis.
3. Next, the DNA is cut into pieces using special enzymes called “restriction enzymes.” These restriction enzymes cut the DNA at specific base pair sequences. Because of the introns and other genetic variations in each individual’s DNA, the resulting DNA fragments are of unique length for each individual.
4. The DNA fragments are separated using gel electrophoresis. The DNA samples are placed in the wells of an electrophoresis gel and separated according to their lengths. Smaller molecules move further and more quickly than larger molecules. (In a typical electrophoresis procedure, the gel would be stained to reveal the location of the separated fragments. In a total DNA fingerprinting procedure, the number of undifferentiated fragments spread over each gel lane would be very difficult to distinguish with the naked eye.)
5. The double-standard DNA fragments that are in the electrophoresis gel are denatured and split into single strands. Heating or treatment with chemicals does this. The gel with the denatured, single-stranded DNA fragments is then applied to a sheet of nitrocellulose paper. It is then heated to permanently attach the DNA to the sheet.
6. The nitrocellulose paper containing the single stranded DNA fragments is treated with a radioactive DNA probe. The probe binds to a complementary sequence in one or more of RFLPs on the nitrocellulose paper. For increased certainty, multiple probes might be used on multiple samples of an individual’s DNA.
7. An X-ray film is placed over the nitrocellulose paper to expose the film at the positions where the probe has hybridized to the complementary DNA sequences in the RFLPs. The resulting banding pattern on the X-ray represents the unique “DNA fingerprint” for the individual. The pattern of two or more DNA samples can then be compared for similarities.

Experiment Overview

A murder has occurred in an apartment building. At the time of the murder, a group of off-duty police officers were watching a football game in a different apartment than where the murder occurred. As soon as the gunshot was heard, the off-duty officers responded by surrounding the apartment building. All individuals within the building were asked to donate a blood sample for DNA fingerprinting. In addition, blood samples were “lifted” from the crime scene. Your job will be to study the background information on DNA structure and the procedures involved in DNA fingerprinting. Then your investigative team will do DNA fingerprints for three suspects to determine if one of them is the murderer. (Remember in the simulation only a small segment of DNA will be used whereas real DNA contains much larger DNA molecules.)

Materials

Safety Precautions

Be careful when handling DNA; it is fragile.

Procedure

Part I. Extraction of DNA

1. Form investigative teams with five pairs of students on a team. Each team pair will be responsible for analyzing one set of DNA. Assign responsibility as follows:

Person 1: Crime scene DNA
Person 2: Victim’s DNA
Person 3: Suspect 1 DNA
Person 4: Suspect 2 DNA
Person 5: Suspect 3 DNA

2. Each pair should use the appropriate pop beads and connectors to assemble their assigned DNA. Assemble the nucleotides from the 3' to 5' strand of DNA first. Then assemble the complementary 5' to 3' strand. Follow the sequence diagrammed on the DNA Sequence Worksheet.

All team members should use the following color scheme:

Adenine—yellow
Cytosine—green
Deoxyribose—white
Guanine—blue
Phosphate—red
Thymine—orange
Connectors—clear

Be sure to keep the 3' to 5' side of the molecules continually oriented on top so that they are all treated in the same manner.

1. For this investigation, two restriction enzymes will be used to cut the DNA into fragments, enzyme NoRo I and enzyme NoRo II. Enzyme NoRoI recognizes the sequence:

3'….ATTA…. 5'
5'….TAAT…. 3'

The NoRoI enzyme then makes the cut in the double-stranded DNA as follows:

….AT TA….
….TA AT….

The NoRoII enzyme recognizes the sequence:

3'….CGGC…. 5'
5'….GCCG…. 3'

And makes the cut:

….CG GC….
….GC CG….

2. Examine the assigned DNA segment and find restriction sites for NoRoI. Look for the exact sites shown in the diagram for NoRoI. Carefully cut (unsnap the connections) the DNA at the restriction sites, keeping the DNA strands properly oriented.
3. Cut the segments further using the restriction sites recognized by the pattern of NoRoII.

Part III. Gel Electrophoresis

1. Locate a Paper Gel Electrophoresis Template.
2. Record the person whose DNA is being analyzed on the top line of the template.
3. Record the number of fragments resulting from the cuts of NoRoI and NoRoII.
4. Simulate gel electrophoresis of the DNA fragments by drawing a bar to represent the resulting DNA fragments on the Paper Gel Electrophoresis Template at the appropriate spot on the mock electrophoresis gel. Do this by counting the number of base pairs in each fragment and drawing a bar representing the fragment on the gel at the appropriate place.

Part IV. Denaturing the DNA into single strands

1. Carefully break the hydrogen bonds that are holding each double stranded fragment together (clear connectors). Remove each 5' to 3' single stranded fragment from each piece of DNA on the gel electrophoresis template.
2. Set the 5' to 3' single strands aside and use them as parts for building probes in Part V.

Part V. Radioactive Probes

1. Use pop beads to assemble three single-stranded probes with a 5'….ATG…. 3' base pair sequence.
2. Use black pop beads instead of red for the phosphate groups. The black beads will simulate and represent radioactive phosphate groups.

3. Move the probe slowly (3' to 5') down the length of each single-stranded fragment. Look for a match for the ATG probe (i.e., a TAC sequence in the 3' to 5' strand). If a match is found where the probe fits in perfect order, attach the probe to the fragment.
4. Add probes to all fragments that contain the proper base pair sequence.

Part VI. Autoradiography

1. When an X-ray film is exposed to the DNA fragments with the radioactive probes, the film will be exposed at the sites of the probes.
2. Locate the Autoradiogram Worksheet and record the location of the radioactive fragments in the proper lane of the worksheet. Do this for the entire analyzed DNA for your investigative team. Do not record the fragments that do not have radioactive probes attached.
3. The Autoradiogram represents each individual DNA segment’s “fingerprint,” so each lane on the autoradiogram represents the DNA fingerprint of each individual.
4. Compare all the DNA fingerprints. Can the guilty suspect be identified from the DNA fingerprints? Answer the following questions on the back of the Autoradiogram worksheet.

1. Based on the autoradiogram’s results, which suspect is the murderer? Explain your answer.
2. Discuss with your investigative team the potentially controversial aspects of DNA fingerprinting. Write a summary of the key discussion points.
3. How might DNA fingerprinting be used to determine if a child belongs to a specific parent? What might a child’s auto-radiogram look like compared to a mother’s and a father’s?

Student Worksheet PDF

10332_Student1.pdf