Where's the CAT?
A DNA Profiling Simulation

By Ellen Mayo Co-Authored By Anthony Bertino

Background Information:

Materials needed:

1. a roll of craft magnet tape, cut into small pieces (16 pieces needed per group)
2. tape (a roll per group for convenience)
3. scissors (at least one pair per group for convenience)
4. envelopes or 2 x 5 inch pieces of white paper
5. brightly colored paper for making the probe
6. photocopies of the DNA sequences
   Click here to download or print the DNA files required for this activity
   
7. a large piece of bulletin board paper (space on a wall or blackboard will do, if things can be taped to it).

The scenario is that a woman was raped and several weeks later finds herself pregnant. She wants to know whether the baby is her husband's child or the child of the suspected rapist. The editorial staff of the journal felt that the topic of rape was too emotional so it was modified to read: The activity simulates the following situation: A married couple (the woman is infertile) arranges with a surrogate to have a baby. The surrogate mother is artificially inseminated with the man's sperm. When the surrogate mother gives birth to the child, she decides that she wants to keep it. She claims that the child's biological father is not the sperm donor, but her own husband. The case is taken to court to decide custody. Genetic testing is done to determine the true biological father.

If you wish to use the above scenario, change all references to "suspect" to read "sperm donor." (Results will be that the husband was the biological father in either scenario.) DNA profiling is now being used in forensic cases as a method of assessing probability of suspect involvement in crimes where DNA samples are available. DNA may be extracted from relatively small samples of cells, such as a blood stain the size of a nickel (about two drops) or a semen stain the size of a dime. When performed under properly controlled conditions and interpreted by an experienced forensic scientist, such profiling can link a suspect to a particular incident with compelling accuracy. This simulation activity allows students to work through the theory of DNA profiling and to grapple with some analytical and ethical questions. It can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. Simpler concepts such as base pairing can also be reinforced as students work through the activity.

DNA profiling involves four basic procedures:

1. Generate DNA fragments using restriction enzymes
O nce the DNA has been isolated from the tissue sample (a procedure not included in this simulation), it is then cut with a restriction enzyme. Such enzymes recognize a specific sequence of bases on the DNA and act as molecular scissors to cut the DNA strand within the recognition sequence. Our enzyme is known as Hae III, which recognizes the sequence GGCC and cuts the DNA between the center G and C, leaving one fragment that ends with GG and another fragment that begins with CC. Within the human genome, this particular sequence will reappear at many points. Because each person inherits a unique combination of sequences, the number and size of fragments created from each person's DNA should be as individualistic as a fingerprint. These different size fragments are the result of restriction fragment length polymorphisms (RFLP's), i.e., within a population, there will be many forms or lengths (polymorphs) in which the restriction fragments may appear as a result of individual differences in the genetic sequences.

2. Separate DNA fragments by gel electrophoresis
The next problem is to separate the fragments according to size. This is accomplished by gel electrophoresis. The digested DNA sample is placed at one end of an agarose gel and electric current is applied. Because DNA is a negatively charged particle, it migrates toward the positive electrode in the gel chamber. The agarose molecules in the gel act as a sieve, allowing the smaller DNA fragments to move through the spaces between agarose molecules faster than the unknowns for comparison and as a check that the gel is uniform and permits each unknown to separate smoothly. At this point if the DNA were stained, there would be so many fragments from each unknown sample that the DNA would appear to be an undifferentiated smear across the gel.

3. Transfer DNA fragments fom the gel matrix
In order to distinguish between the DNA of one individual and that of another, there must be a way to identify a specific pattern of inherited alleles. In an attempt to solve this problem, the DNA smear is transferred to a nylon filter to prevent further movement and degradation of the DNA fragments. In the process of transferring the DNA from the gel to the nylon filter, the double strands are separated, and the filter contains only single stranded DNA, to which a complementary sequence of DNA could bind. (NOTE: This simulation uses only a single strand of DNA throughout the restriction analysis procedure for purposes of simplification.)

4. Radioactive DNA hybridization
Radioactively labeled sequences of single strand DNA have been developed that recognize particular sequences of DNA that are not thought to be transcribed, but which do appear in a randomly repeated fashion throughout the genome. These fragments are called variable number tandem repeats (VNTR's); these fragments have been developed to identify particular sets of alleles, and extensive work is now being done to quantify the frequency of particular VNTR's within a population. Each individual should inherit one allele of a particular size and containing a VNTR from one parent and a homologous but not necessarily identical allele with the same VNTR from the other parent. If several labeled DNA strands of particular sequences (called probes) are allowed to combine with the DNA sample of the filter, one probe at a time, the resulting combinations of labeled fragments should be able to distinguish the DNA of one individual from that of another.

Procedure:

Each group of 2-4 students should be assigned to one sample. A minimum of five groups can be assigned. Students can work separately if materials and time permit. To make the simulation manageable, each simulation base represents approximately one thousand bases (one kilobase) that would be found in an actual DNA profile. To simulate the restriction digest, each group must first cut out the strips of DNA sequences, then tape together the strips representing one sample of DNA, being sure to match and obscure the subscripts as the sample is assembled. Next, scan the sample strip for the probe sites: CAT. Wherever the sequence CAT appears, tape a 1 cm piece of magnet to the back of the strip. Next, mark the sample strip at the recognition sites for the restriction enzyme Hae III (GGCC). Then cut the strip all the way across between the center G and C of each restriction site. (NOTE for teachers: Each group with a sample should have six smaller fragments at this point; the standard should yield eight smaller fragments. Provide each group with a small labeled envelope. This can represent a well in the agarose gel.)

To simulate gel electrophoresis, use a large (at least 62 cm x 74 cm) sheet of poster board, preferably in a contrasting color, on which to assemble the fragments resulting from the restriction digest. The standard should be placed first. Exact distances from the origin in the "well" are not important, as long as all fragments of the same length are placed the same distance from the well, and the larger fragments are placed closest to the well with the smaller ones being placed farther away in descending order beneath the well. Use the envelope that contained the standard fragments to represent the well. Place the 20-base fragment at least 5 cm below the well, the 18-base fragment at least 5 cm from the first fragment, and so on until all eight fragments have been distributed down the column. Tape the fragments in place. Place the envelope for the mother's sample to the right of the standard sample envelope, then repeat the placement of fragments in another lane parallel to the standard lane. Note that the mother's 20-base fragment should be the same distance from its well as the standard 20-base fragment is from its well. The mother's DNA yields a 9-base fragment; this should be placed at a distance intermediate to the 10- and 8- fragments of the standard. Continue placement of samples in the same manner, moving to the right across the poster board in the following order: husband, suspect, and child. When complete, each sample contains six different fragments. The fragments of one sample are the same size as the fragments of the other samples, but they differ from those of the other samples in their specific base sequences.

These fragments must next be differentiated from one another; the sequences in a real gel would not be known. Construct DNA probes by cutting 1 cm by 2 cm rectangles from the brightly colored construction paper. The standard group will need eight rectangles and the sample groups will need two each, for a total of sixteen rectangles needed for the class. The sequence "GTA" should be written on each rectangle (a template for photocopying is included) and a 1 cm magnet should be taped to the back of each. These DNA probes will be used to identify specific alleles. The probe sequence (GTA) is complementary to the VNTR sequences (CAT). With a probe in hand, scan the standard and position a GTA probe on each VNTR site. Notice that each fragment in the standard has a VNTR site and can be labeled with the probe. (This procedure of combining DNA from two sources is called hybridization.)

Repeat this procedure for each sample. Notice that only two fragments from each sample will bind with the probe. Each labeled fragment represents a part of one chromosome of a homologous pair. Analyze the banding patterns according to Mendelian principles : the child inherited one allele from each parent. In our example, the mother could only have donated one the child's labeled alleles. Which man is more likely to have donated the other allele? It helps students to visualize this relationship if all sequences which do not bind to the probe are removed from the "gel" so that only the probed sequences will be compared. This is what an autoradiograph would look like.

Further discussion could deal with various situations. What if the biological father were the rapist? Would abortion be justified here? What if the samples represented two suspects in the rape instead of the husband and suspect, and a stain from the woman's clothing instead of a fetus? Would this be sufficient evidence to convict a suspect of the crime? What limitations can be seen in these procedures?

Other concepts that can be added include the reading of DNA sequences from 5' to 3' ends, the use of multi-locus probes, nucleotide structure (as the production of labeled probes is discussed), and the use of other restriction endonucleases. Other situations could be developed if a rape is inappropriate for discussion. For example, there is a British immigration case where a child was temporarily denied admission to Great Britain from Ghana until it was shown by DNA technology that he was, indeed, the child of his mother rather than of his aunt as was alleged.

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