Who’s the Daddy? Using DNA Profiling to Determine Paternity
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.
- DNA profiling
- DNA structure and base pairing
- Restriction enzymes
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:
- 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.
- 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.
- 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.
- 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.
- 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.
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.
Highlighter pen (optional)
Magnetic tape, 4 cm
“Radioactive probe” locators
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:
- When making a real DNA profile, what are the molecular “scissors” used to cut up the DNA strands?
- Inside what kinds of organisms were these molecular “scissors” first discovered? For what purpose were they used?
- Use a dictionary or textbook to define palindrome and write a word or phrase that would be considered a palindrome.
- Since both men in the story have the defective gene, what does that mean for Rachel?
- In the absence of scientific evidence, could the deceased man be Rachel’s father? Explain your answer.
Although the materials in this activity are considered nonhazardous, please follow all normal laboratory safety procedures, especially when using scissors.
Step 1. DNA Isolation and Purification
For this simulation, the “DNA samples” have already been isolated but they must have the “impurities” removed.
Step 2. Simulated Restriction Enzyme Digestion
- Obtain one page of an isolated “DNA sample” from the instructor.
- 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!
- 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 3. Simulated Gel Electrophoresis
- 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.)
- Count the number of base pairs in each RFLP and write the number in the upper left corner of the fragment.
- 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.
In the area designated by your instructor, attach the RFLPs made in step 1 according to the following instructions:
Step 4. Simulated Southern Blot
- 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.
- 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.
- 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.”
- 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.
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.
Step 5. Simulated Detection and Analysis of Target DNA Fragments
- 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.
- 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.
- 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.”
- 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.”
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.
- Prepare your group’s part of the autoradiograph by cutting apart the four GTA rectangles you have received.
- 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.
- 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.
- 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.
- 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.