Materials Included In Kit

Safety Precautions

Although the materials in this activity are considered nonhazardous, please follow all normal laboratory safety procedures, especially when using scissors.

Disposal

Since the GTA rectangles may be reused, have students carefully remove them from the “gel,” place them in the group’s zipper-lock bag and return them to you for storage. The remaining RFLPs may be removed and thrown in the trash.

Lab Hints

• Enough materials are provided in this kit for 30 students working in groups. All materials are reusable. This laboratory activity can reasonably be completed in one or two 50-minute class periods depending on the level of your students.
• Prior to the activity, make a copy of the “Radioactive Probe” Locators page on the brightest, colored paper available (e.g., hot pink, lime green). Cut into horizontal strips of four GTA per strip.
• Prior to the activity, make enough copies of the Electrophoresis Gel “Blotter” page for each group member.
• Feel free to change the Prelab Activity scenario if the one provided is not understandable or a more CSI-type scenario is desired.
• If students are cutting apart and assembling the DNA segments when the class period ends, place the cut and uncut pieces inside labeled zipper-lock bags until the next day.
• The staples, representing the radioactive probes, must be steel, not aluminum, in order for the magnetic tape on the back of the “probe” locators to stick.
• To prevent the same DNA segments from always being the “mom” or “dad,” rearrange the parent sequences to represent different persons in the story according to the following:

  —Parent A should always be the “father” that does not match.
  —Parent B and Parent C could be either a mother or a father.
  —Parent D could be used as either a mother or father but not with Parent B.

• The child sequence, which in this scenario would be Rachel, and the standard sequences should not change.

Teacher Tips

• Use the set of six simulated DNA sequences included in the kit as the master copies from which student copies are made.
• Depending on the level of your students, you may want to read the Background and Prelab Activity scenario out loud, as a class, and then have students answer the questions as a group.
• Although written as an activity for the entire class to do together, feel free to make smaller groups and give each group a complete set of five simulated DNA sequences to prepare according to the instructions. After all the groups have laid out the fragments on a desktop or tabletop and have completed steps 5–16, you may want to finish the activity as a class. Assign different groups to place one set of RFLPs on the wall/chalkboard and then add the probe locators.
• Students should understand that in real DNA samples, the letters representing the nitrogen bases are not visible and actual RFLPs would be several thousand base pairs long.
• The technique referred to as a Southern Blot was named after Edward Southern, who first developed the process at Edinburgh University in Scotland in the 1970s.
• Unknown RFLP sizes (lengths) are determined by running a known set of DNA fragment lengths, cut with the same restriction enzyme, alongside a set of unknowns. By comparing how far the unknown fragments traveled relative to the known fragments, the size (length) of the unknown fragments can be determined. Fragments are always measured in Kilobases or KB. 1 Kb = 1000 bp (base pairs).
• The C.A.T. sequence on the DNA RFLPs, to which the “radioactive probes” attached, is a very simplistic example of a VNTR or Variable Number Tandem Repeat. Most VNTRs—sometimes called “junk DNA” because they are not known to code for any type of protein—used in forensics consist of longer DNA sequences (up to 70 base pairs).
• Two DNA Parentage test reports and the explanation for their interpretation are included in the kit. This information was provided by a certified and accredited DNA diagnostic testing center. Transparencies and/or copies may be made and used in a class discussion. This material would be appropriate in directing a discussion of the answer to Post-Lab Analysis Question 13.
• Use the Parentage Test Reports to also point out the fact that, unlike this simulation, a real DNA profile of multiple individuals would not contain as many RFLPs with identical sizes.

Answers to Prelab Questions

1. 1. When making a real DNA profile, what are the molecular “scissors” used to cut up the DNA strands?

      Restriction enzymes cut up DNA into fragments.

   2. Inside what kinds of organisms were these molecular “scissors” first discovered? For what purpose were they used?

      Restriction enzymes were found to be present in bacteria. The enzymes protect the bacteria from infection by viral DNA.

2. Use a dictionary or textbook to define palindrome and write a word or phrase that would be considered a palindrome.

   A palindrome is a word or phrase that is spelled the same backward and forward (i.e., mom, dad, racecar, madam I’m adam).

3. Since both men in the story have the defective gene, what does that mean for Rachel?

   It means that she could have inherited the defective gene from either man.

4. In the absence of scientific evidence, could the deceased man be Rachel’s father? Explain your answer.

   Yes, if the mother was in the very early stages of pregnancy, but didn’t know it, when her first husband died.

Sample Data

Student data will vary if other parent combinations are used.

Answers to Questions

1. Before a DNA profile can be made, what must a scientist be able to obtain from the organisms involved in the profile?

   A tissue sample that contains DNA.

2. Using restriction enzymes to cut DNA strands into RFLPs is referred to as “digestion.” Why?

   It is referred to as digestion because the DNA is cut up into smaller pieces by the restriction enzymes just as the proteins, fats and carbohydrates in food are “cut” into smaller pieces by enzymes during digestion.

3. If the DNA samples used in this activity were real, how many base pairs did the longest and shortest RFLPs have? [Express your answer according to the formula: 1 letter = 1 KB = 1000 bases]

   In this simulation, each letter represents 1,000 base pairs. The longest RFLP with 20 KB would contain 20,000 bases. The shortest RFLP with 6 KB, would contain 6,000 bases.

4. If another type of molecular “scissors” had been used in this activity, do you think the RFLPs would have been the same lengths or different? Explain your answer.

   The RFLPs would be different lengths because each restriction enzyme cuts DNA only at its specific palindrome “recognition site.” Where an enzyme’s recognition site is located and how many of them are present in the DNA sample determines the length and the number of fragments obtained.

5. Why is it necessary to make and run a standard set of RFLPs, cut from DNA with the same molecular “scissors,” alongside the RFLPs of the other DNA samples?

   The standard is important for two reasons: First, to ensure that the gel has a good consistency, without lumps, etc. Second, running DNA fragments of known lengths alongside unknown fragments helps in determining the lengths of the unknown fragments.

6. Why is the technique referred to as a Southern Blot such a critical part of this DNA profiling process? In other words, when the Southern Blot step is complete, what is it supposed to accomplish?

   This process is designed to allow one to visualize specific groups of macromolecules, in this case, specific target DNA fragments useful in identifying the real father.

7. What was done in this activity to simulate the three major parts of the Southern Blot process? Be specific!

   To simulate the denaturing of DNA to produce single strands, the bottom half of each RFLP was cut off. To simulate the transfer of the RFLPs to a nylon membrane, a drawing was made of the classroom “gel” indicating the location of each RFLP. To simulate the attachment of radioactive probes to VNTRs on the RFLPs, staples were added.

8. 1. What was the target VNTR sequence to which the radioactive probes attached themselves?

      C.A.T.

   2. What was used to simulate the attachment of the probes to the VNTR sequence identified in Question 8a?

      Two staples were added to each C.A.T. sequence on every RFLP that had a C.A.T. sequence.

9. In this simulation, how was the location of the radioactive probes on the RFLPs detected?

   Brightly colored rectangles with the complementary code—G T A—were attached with magnets to the “radioactive probes” (the staples) on each C.A.T. sequence.

10. 1. From the evidence presented, who was Rachel’s real father?

       Student answers will vary depending on how the scenario is set up.

    2. Briefly explain the evidence supporting your answer to Question 10a? [The explanation should include something about the size (length) of the RFLPs.]

       Children inherit half of their alleles (genes) from one parent and the other half from the other parent. After making the “autoradiograph” with the bright colored rectangles, it was obvious which target RFLPs Rachel had inherited from her parents; there was no reasonable doubt.

    3. At which step in the profile-making process was the identity of the father overwhelmingly in favor of one man over the other?

       Student answers will vary but should include something like, “When the autoradiograph was made in step 5—Detection and Analysis of Target DNA Fragments—there was no question as to who Rachel’s father was.”

    4. Why would it have been difficult to determine the real father prior to the step listed as your answer to Question 10c?

       Prior to making the “autoradiograph,” the target DNA fragments containing the radioactive probes, necessary for identifying the fragments, were not visible.

11. Besides the huge size, how else do you think the autoradiograph made in class differs from one that would be made in a DNA forensics or paternity-testing laboratory?

    The autoradiograph made in class used brightly colored paper to identify the location of the radioactive probes. A real autoradiograph uses X-ray film and, like X-rays, everything would appear black and white.

12. List at least three sources of error that could affect the outcome of any DNA profile.
    1. The DNA is not processed soon enough and becomes too degraded to use.
    2. Lab technicians may not have followed the procedures exactly.
    3. The DNA may have been “cut” with different restriction enzymes making a comparison impossible.
    4. A standard set of DNA fragments is not run and therefore there is no way to know if the gel had lumps or other inconsistencies.
13. If a lawsuit for non-payment of child support was filed against the person listed as your answer to Question 10a, based on the evidence presented, should this person be required to pay? Why or why not?

    Student answers will vary but may include that based solely on the evidence in this activity, the answer is No! With only one matching fragment, there is a strong probability that many other people in the world may have that same matching fragment. Therefore, the suspect could not be required to pay because there is reasonable doubt that he is the real father.

14. Make a vertical copy of the following flow chart below on your answer sheet. (a) Fill in the blanks in front of each partial statement with a verb(s) that describes each step of the DNA fingerprinting process. (b) Which step(s) in the flow chart are needed to complete a Southern Blot?
    {10678_Answers_Figure_4}

Teacher Handouts

10678_Teacher1.pdf

References

The Science Teacher, NSTA, December, 1991.

Where’s the CAT? A DNA Profiling Simulation. http://www.accessexcellence.org/AE/AEC/AEF/1995/mayo_dna.html (Accessed May 2018)

Introduction

Is there any way to prove that a relationship exists between two organisms? In humans, blood typing has been used for many years to help determine relationships but in many cases, this technique may not be definitive. How does one determine if a relationship exists between two snow leopards living in different zoos or between two iguanas living on separate islands? The technique of DNA profiling has provided answers to many similar questions since it was first developed in the mid-1980s.

Concepts

• DNA profiling
• Palindromes
• DNA structure and base pairing
• Restriction enzymes

Background

DNA profiling or DNA fingerprinting was developed by a British researcher, Alec Jeffries, in 1984. He is credited with being the first person to solve immigration, paternity and murder cases using this technique. The uses for this powerful technique have now expanded to include diagnosing genetic diseases, identifying victims of disasters, solving cases of rape, identifying endangered plants and animals, and establishing evolutionary relationships among different organisms. Every organism carries a unique combination of DNA patterns that provides virtually irrefutable identification.

Two significant discoveries were necessary to make DNA profiling possible. First, it was found that about 30% of DNA consists of relatively short (4–8 nitrogen base pairs), highly repetitive sequences which vary significantly from one organism to another. Secondly, in 1962 enzymes were discovered that “restrict” or cut up the DNA of viruses (called bacteriophage) that attack bacteria. The most useful restriction enzymes were found to always cut DNA at the exact same place every time—at specific palindrome sequences. For example, the restriction enzyme HaeIII always cuts DNA between the middle G and the middle C of this palindrome:

Another enzyme, called EcoRI, cuts DNA between the G and the A in this palindrome: Since the DNA of all organisms contain the same nitrogen bases and the aforementioned repetitive sequences are found in all DNA, restriction enzymes could be used to slice up the DNA from any organism.

DNA profiling became even more useful and powerful with the development of the process known as Polymerase Chain Reaction (PCR). Using PCR, minute samples of DNA no larger than the head of a pin can be multiplied to produce billions of copies in a matter of hours.

There are five basic steps to creating a DNA profile of this type:

1. DNA Isolation and Purification: DNA is obtained from the cells of any tissue sample (e.g., bone, blood, roots of hair, skin cells, saliva), and impurities are removed. If only a very small sample of tissue is available, the DNA from the sample may be subjected to PCR to increase the amount of DNA.
2. Restriction Enzyme Digestion: After the DNA has been purified, restriction enzymes are added to the sample. The enzymes cut the DNA into fragments of varying lengths.
3. Gel Electrophoresis: The digested DNA sample is then stained with a dye and placed into the wells (holes) of an agarose gel in an electrophoresis chamber. When the electricity is turned on, the DNA fragments migrate through the gel according to their size, with smaller fragments moving faster than larger ones.
4. Southern Blot: When electrophoresis is finished, the gel is removed from the chamber and soaked for 45 minutes in a basic (pH) solution that denatures the DNA. The double-stranded DNA separates into single strands, which makes them easier to analyze. The single-stranded DNA is then transferred to an inert nylon membrane in a process known as blotting. By simple capillary action, the DNA fragments are transferred to the membrane in exactly the same pattern and location as they existed in the gel. The final step of the process involves immersing the membrane in a solution containing small radioactive DNA sequences called “probes.” These specially made probes are designed to attach only to complementary sequences on the DNA fragments called VNTRs or Variable Number Tandem Repeats. For instance, if the probe has the sequence A-T-C, it will attach only to a T-A-G VNTR sequence on the DNA fragment.
5. Detection and Analysis of Target DNA Fragments: After overnight incubation with the radioactive probes, the membrane is washed again to remove any unbound probes and then placed on top of a piece of X-ray film. When developed, black spots appear on the film wherever a radioactive probe was joined to its complementary DNA sequence. These X-ray “pictures” of DNA fragments are known as autoradiographs or autorads.

Classic crime-fighting evidence, like fingerprints and blood types, can only be used to prove that a suspect was not involved in a specific activity and is therefore referred to as “exclusionary evidence.” In contrast, DNA profiling can identify the suspect(s) that was involved, regardless of alibis or witnesses to the contrary.

Experiment Overview

The purpose of this activity is to simulate the creation of a DNA profile. Each of the five steps of the process will be simulated and the resulting profile used to determine the paternity (father) of an individual.

Materials

Prelab Questions

Prelab Activity

Read the following scenario, and answer the questions that follow on a separate sheet of paper. This was a real medical scenario, but the names have been changed.

A pregnant young woman, named Rachel, comes into a hospital emergency room experiencing severe labor pains and is quickly admitted. Later that afternoon, with her husband nearby, she gives birth to an apparently healthy baby boy; they name him John Michael.

The first 48 hours pass without incident. However, as a nurse prepares the baby to leave the hospital, his loud crying and movements indicate more than a minor problem. When the pediatrician is notified, she orders an upper GI (gastrointestinal) test and a blood test. The upper GI test reveals a blockage of the baby’s small intestine and the results of the blood test reveal elevated levels of a protein normally present in much lower amounts. The pediatrician informs the parents that surgery will be necessary to remove the intestinal blockage. In addition, the blood test strongly suggests that John Michael has inherited cystic fibrosis (CF), a genetic defect in which only 10% of affected children live to the age of 30.

Shocked by the news, Rachel and her husband cannot understand how this could have happened. Cystic fibrosis is always inherited as an autosomal recessive genetic trait, meaning that both parents would have to be carriers of the trait. However, as carriers, the parents have a 25% chance of passing the defect on to their offspring. Is it possible that two people, from completely different backgrounds, can be carriers of the same defect?

When Rachel’s husband calls his dad, he discovers that the only sibling, a brother, of his deceased mother died of CF at the age of 22. Rachel’s husband must have inherited the defective gene from his mother.

What about Rachel? As the oldest child in her family she never knew her mother’s first husband, although she had seen pictures of him taken on their wedding day. Unfortunately, he was killed in a one-car rollover accident on their honeymoon. Needing support and comfort, Rachel’s mom had married a childhood friend shortly after the funeral. While discussing John Michael’s diagnosis, Rachel learns that no history of CF exists on her mother’s side of the family; the defective gene must have come from her father. But, is her father the man she has always known as “daddy” or the man smiling at her from old wedding pictures?

Rachel’s mother has her first husband’s body exhumed and several tissue samples are taken. Although recently divorced from her mother, Rachel’s father—Jim—consents to donate tissue for both genetic screening and parentage testing. When the genetic screening tests are completed, both men are found to possess the defective CFTR (cystic fibrosis transmembrane conductance regulator) gene on Chromosome 7. Is it possible for Rachel to know for sure who her father is?

Use the background information and the scenario to answer these questions:

1. 1. When making a real DNA profile, what are the molecular “scissors” used to cut up the DNA strands?
   2. Inside what kinds of organisms were these molecular “scissors” first discovered? For what purpose were they used?
2. Use a dictionary or textbook to define palindrome and write a word or phrase that would be considered a palindrome.
3. Since both men in the story have the defective gene, what does that mean for Rachel?
4. In the absence of scientific evidence, could the deceased man be Rachel’s father? Explain your answer.

Safety Precautions

Although the materials in this activity are considered nonhazardous, please follow all normal laboratory safety procedures, especially when using scissors.

Procedure

Step 1. DNA Isolation and Purification

For this simulation, the “DNA samples” have already been isolated but they must have the “impurities” removed.

1. Obtain one page of an isolated “DNA sample” from the instructor.
2. Use scissors to separate each of the double-stranded DNA segments on the page and trim the edges. Caution: Do not cut off the asterisk(s) on the right side!
3. Assemble the separated DNA segments into one long simulated DNA molecule by placing the left edge of the second strand over the asterisk at the right edge of the first strand. Secure the two strands with a piece of clear tape. Follow the same pattern to assemble the rest of the strand. Continue adding each DNA segment in turn until the entire DNA molecule is assembled. Note: The assembled strand is correct if a 5′/3′ end is visible on the left and a 3′/5′ end is visible on the right.

Step 2. Simulated Restriction Enzyme Digestion

4. Scan the entire length of the DNA molecule and find all the GGCC sequences, reading from left to right. Hint: The sequence on the opposite (antiparallel) strand should read CCGG because the two sequences together make a palindrome. Use scissors to cut vertically through the DNA molecule between the middle G and C of every sequence. (This cutting simulates using the 4-cutter restriction enzyme HaeIII. If done correctly, there will be six fragments, called Restriction Fragment Lengths Polymorphisms or RFLPs. The group with the “standard” DNA molecule will have eight fragments.)
5. Count the number of base pairs in each RFLP and write the number in the upper left corner of the fragment.
6. Put all the RFLPs from your DNA molecule into a zipper-lock bag labeled with the name of the group’s sample (i.e., Standard, Child, Mother), according to your instructor’s direction.

Step 3. Simulated Gel Electrophoresis

In the area designated by your instructor, attach the RFLPs made in step 1 according to the following instructions:

7. The group with the “Standard” DNA will place their RFLPs first. Remove all RFLPs from the bag and attach the labeled bag at the top of the “gel” (e.g., wall, poster board, wallboard) with tape, etc. Note: The bag will represent the “well” into which real DNA RFLPs would be loaded.
8. Place clear tape on the top half of each RFLP. Beginning with the longest (the one with the most base pairs), use a meter stick to measure and attach it 5 cm below the bag. Place the other RFLPs by length, 5 cm below the preceding ones.
9. After each RFLP has been placed on the classroom “gel,” write the number of base pairs (their length) on the “gel” so the other student groups can see the number when placing their own RFLPs. Note: Fragments should be in descending order with the longer ones closest to the “wells.”
10. The group that has the DNA RFLPs from the “mother,” as indicated by the instructor, will follow the same procedures as the first group. Attach the bag and use a meter stick to help align the RFLPs vertically and horizontally with the standard RFLPs. Note: The RFLPs of the two potential fathers should be added next and the child’s RFLPs last.

Step 4. Simulated Southern Blot

In this step, the denaturing of DNA from double to single strands, the transfer of DNA RFLPs to a nylon membrane, and the addition and attaching of radioactive probes to complementary DNA sequences will be simulated.

11. Two members of each group return to the classroom “gel” and mark the location of each RFLP. Remove it from the “gel” and use a pair of scissors to cut off the bottom half (the complementary strand) of each RFLP.
12. When all the RFLPs for the standard and every individual have been “denatured” to single strands and put back in the “gel,” use the Electrophoresis Gel “Blotter” page to draw lines showing the exact location of all DNA fragment lengths for each individual.
13. Have two members of the group return again to the classroom “gel.” Scan the group’s RFLPs, locate every base sequence that spells C.A.T. and put a small checkmark on the “A.”
14. While one person marks the location on the “gel,” remove only the checked RFLPs. Use a stapler to place one staple horizontally through the “A.” Return these RFLPs to their designated places on the classroom “gel.”

Step 5. Simulated Detection and Analysis of Target DNA Fragments

In the preparation of a real DNA fingerprint, even after a 24-hour incubation, the exact location of the radioactive probes is not apparent. To find where each probe is attached and locate the DNA fragments of interest, the membrane is laid on top of a piece of X-ray film for 18 to 72 hours. When the film is developed, the target DNA fragments’ location and size are revealed because the attached radioactive probes make only those fragments visible. This process is called autoradiography.

15. Prepare your group’s part of the autoradiograph by cutting apart the four GTA rectangles you have received.
16. Obtain a length of magnetic tape and cut four 1-cm pieces. Remove the paper backing, and stick one piece to the back of each GTA rectangle.
17. Keep two of the GTA rectangles for the group and give the rest to the instructor. Note: The DNA “standard” group will keep all four GTA rectangles and get four more from the instructor for a total of eight.
18. Have a person from the group return to the “gel” with the prepared radioactive probe locators. Find the sequence on the RFLPs that is complementary to the sequence on the probe locators. Attach the rectangles, then remove all unmarked RFLPs, throw them away, and be seated.
19. On each person’s Electrophoresis Gel “Blotter” page, highlight or circle the RFLPs on the diagram that have GTA sequences attached. As a group, answer the Post-Lab Analysis Questions.

Student Worksheet PDF

10678_Student1.pdf