In this activity, freshman college students learn biotechnology techniques while playing the role of a laboratory technician. They perform simulations of three diagnostic tests used to screen newborns for cystic fibrosis. By performing an ELISA, a PCR analysis, and a conductivity test, students learn how biotechnology techniques can be used to diagnose diseases. Students get excited when they realize they will be performing tests actually used in a real-life situation. This exercise is also appropriate for high school students.

Biological concepts such as atoms and ions, channel proteins, chromosomes, genetic mutations, protein modification, classical genetics, and evolution can all be taught using the disease cystic fibrosis (CF) as an example. Using a single disease like CF provides a constant thread linking these seemingly unrelated topics. The “Screening Newborns for Cystic Fibrosis” laboratory exercise, designed to take place at the end of the semester, allows students to apply the numerous biological concepts they have been learning throughout the semester. In addition, students complete a prelab WebQuest (Table 1) on CF that reinforces the concepts they have learned and provides additional background knowledge for the exercise. Students visit preselected websites and answer questions about CF, then bring their answers to lab to share with their lab partners. Once this is completed, they are ready to take on the role of lab technician and screen imaginary newborns for the disease.

Table 1.

Prelab activity.

A Cystic Fibrosis WebQuest SiteQuestions
An Introduction to Cystic Fibrosis: http://www.nhlbi.nih.gov/health/dci/Diseases/cf/cf_what.html 
  • What is cystic fibrosis (CF)?

  • How is it inherited?

  • What are the symptoms?

 
The CF Protein: Section 1 – Understanding the Problem: http://learn.genetics.utah.edu/content/genetherapy/gtintro/understandingtheproblem2.swf 
  • What is the normal role of the CF protein?

  • What happens when the protein does not work properly?

 
Screening Newborns: http://www.cff.org/AboutCF/Testing/NewbornScreening/ 
  • What are the advantages of screening newborns?

  • What, if any, are the disadvantages?

  • Do newborns in Connecticut get screened for CF?

 
Immunoreactive Trypsinogen Test: http://www.labtestsonline.org/understanding/analytes/trypsinogen/glance.html 
  • Why is further testing needed if the trypsinogen test is positive?

 
Genetic Testing: http://www.labtestsonline.org/understanding/analytes/cf_gene/glance.html 
  • What chromosome is the CF gene on?

  • What type of mutation is Δ508?

 
Sweat Test: http://www.labtestsonline.org/understanding/analytes/sweat_chloride/glance.html 
  • Why do you think the sweat test is considered the confirming test for CF?

 
A Cystic Fibrosis WebQuest SiteQuestions
An Introduction to Cystic Fibrosis: http://www.nhlbi.nih.gov/health/dci/Diseases/cf/cf_what.html 
  • What is cystic fibrosis (CF)?

  • How is it inherited?

  • What are the symptoms?

 
The CF Protein: Section 1 – Understanding the Problem: http://learn.genetics.utah.edu/content/genetherapy/gtintro/understandingtheproblem2.swf 
  • What is the normal role of the CF protein?

  • What happens when the protein does not work properly?

 
Screening Newborns: http://www.cff.org/AboutCF/Testing/NewbornScreening/ 
  • What are the advantages of screening newborns?

  • What, if any, are the disadvantages?

  • Do newborns in Connecticut get screened for CF?

 
Immunoreactive Trypsinogen Test: http://www.labtestsonline.org/understanding/analytes/trypsinogen/glance.html 
  • Why is further testing needed if the trypsinogen test is positive?

 
Genetic Testing: http://www.labtestsonline.org/understanding/analytes/cf_gene/glance.html 
  • What chromosome is the CF gene on?

  • What type of mutation is Δ508?

 
Sweat Test: http://www.labtestsonline.org/understanding/analytes/sweat_chloride/glance.html 
  • Why do you think the sweat test is considered the confirming test for CF?

 

Working in groups of four, students put on their gloves and goggles and pick up their diagnostic packets. They receive Guthrie cards with “blood” samples from four newborns. The “blood” is actually red marker, and all the tests are simulations. After recording the patient information from the cards, students punch out a circle of “blood” and begin their tests. The first test is for immunoreactive trypsinogen (IRT) and is performed using an ELISA kit. Trypsinogen is produced in the pancreas and travels through ducts to the intestine, where it is converted to trypsin (Bowling & Brown, 1988). Because of the thick mucus produced in the pancreatic ducts of patients with CF, the trypsinogen is not readily converted to trypsin, leaving the levels of trypsinogen in the blood high enough to be detected by an ELISA (Bowling & Brown, 1988). The students perform the ELISA reactions and identify the patients that have positive results. They then refer to the flow chart in their diagnostic packet to determine the next screening test to perform.

The next test is a polymerase chain reaction (PCR) analysis to determine the genotype of the patient. Students perform this test on the patients they have previously identified as having a high trypsinogen level. Approximately 70% of the patients with CF have a three- base-pair deletion in the gene that codes for the cystic fibrosis conductance transmembrane regulator (CFTR) protein (Lap-Chee, 1995). Students perform the PCR analysis, run an electrophoresis gel, and analyze the results. The simulation exaggerates the differences in gene size so that students can readily identify the high and low molecular weight band on the gel. They then interpret their results, refer back to their diagnostic packet, and determine what test to perform next.

The final test that students perform is the sweat test, which is a confirmatory test for CF. This is considered a confirmatory test because it tests for a physical phenotype associated with the disease. The sweat test is performed using a simulated sample of sweat from the patient and a conductivity meter. Patients with CF have more chloride ions in their sweat than normal, so their sweat is a better conductor of electricity (Cystic Fibrosis Foundation [CFF], 2010).

Upon completion of the tests, students work through a series of questions with their lab partners. This allows them to talk about the tests they performed and reflect on what they learned. Finally, the students are asked to write a letter to the parents of the patient(s) that tested positive for CF. In the letter, they explain the screening process and the results as well as provide information about CF. Students enjoy writing the letter; it provides a creative way to express their understanding of the concepts and techniques. As they explain the disease and the diagnosis process to the parents of the patients, it is clear that the students gained a more thorough understanding of mutations, inheritance, and biotechnology.

Prelab Activity

Complete the WebQuest (Table 1) before beginning the lab exercise, and write the answers in your lab notebook.

Laboratory Activity: Screening Newborns for Cystic Fibrosis

Background: You are working as a medical technologist in the Laboratory Medicine Department of a local hospital. One of your jobs involves screening newborns for cystic fibrosis. The screening process is outlined in Figure 1. Each morning, you receive approximately 4 blood-card samples. You put on your goggles and gloves and retrieve the samples from the laboratory supervisor. These samples were obtained by sticking the heel of the newborn and collecting blood on a Guthrie card. Record any pertinent information from the Guthrie card in your lab notebook.

Figure 1.

Flowchart of the process for screening newborns for cystic fibrosis.

Figure 1.

Flowchart of the process for screening newborns for cystic fibrosis.

  • Patient 1 History: Baby Boy Frederick Star born November 2, 2013. Blood sample obtained and forwarded to lab. I.D. 249964-3. Mother: Philomena Star.

  • Patient 2 History: Baby Girl Happy Azaclam born November 2, 2013. Blood sample obtained and forwarded to lab. I.D. 249964-4. Mother: Mary Azaclam.

  • Patient 3 History: Baby Boy Sam Tider born November 2, 2013. Blood sample obtained and forwarded to lab. I.D. 249964-2. Mother: Ethel Tider.

  • Patient 4 History: Baby Girl Lily Root born November 2, 2013. Blood sample obtained and forwarded to lab. I.D. 249964-1. Mother: Rosie Root.

Sample Preparation

Using the paper punch, carefully punch a circle from the Guthrie card for patient 1, cut the small punch circle into four pieces, and place them on the appropriate card. Repeat for samples 2–4.

First Screening Test: Immunoreactive Trypsinogen in Whole Blood ELISA

You will perform a chromogenic (color change) ELISA test for immunoreactive trypsinogen (IRT) as the initial test to screen newborns for CF.

Materials (per group):

  • Four microcentrifuge tubes, each containing extraction buffer and labeled 1–4

  • Positive control sample

  • Negative control sample

  • A strip of eight wells

  • Primary antibody

  • Secondary antibody

  • Substrate/chromogen

  • Transfer pipettes

Procedure:

  1. Place one-quarter of the paper punch from patient 1 into tube 1 containing the extraction buffer.

  2. Repeat for samples 2–4.

  3. Let sit for 5 minutes. Read the rest of the protocol while you wait.

  4. Place 3 drops of the extraction buffer from tube 1 into a well labeled “1”.

  5. Repeat for samples 2–4 and for the positive and negative control samples.

  6. Add 3 drops of primary antibody to each well.

  7. Add 3 drops of secondary antibody to each well.

  8. Add 3 drops of chromogen/substrate to each well.

  9. Incubate at room temperature for 5 minutes and examine the wells for a color change. If the color is not visible or faint, incubate for an additional 5 minutes.

  10. Record your results in the chart below.

Reference data: Check your patients’ results against the following reference data and your controls.

  • IRT level <70ng/mL = no color change (or light green) = Normal levels of IRT detected

  • IRT level ≥70ng/mL = color change (purple) = Abnormal levels of IRT detected

IRT ELISA results:

SampleResult (well color)Interpretation
Positive control   
Negative control   
Patient 1   
Patient 2   
Patient 3   
Patient 4   
SampleResult (well color)Interpretation
Positive control   
Negative control   
Patient 1   
Patient 2   
Patient 3   
Patient 4   

Analysis of data and recommendations:

  1. Which of the patients have a normal IRT level?

  2. Which have a high IRT level?

  3. Does a high IRT level mean that the patient has cystic fibrosis?

  4. Refer to the diagnostic flow chart – which patients should be tested to determine their genotype?

Second Screening Test: PCR Detection of the Cystic Fibrosis Gene Mutation

In this next test, you will amplify the CFTR gene from those patients who had a positive IRT test. The genetic mutation can be detected using a polymerase chain reaction (PCR). The amplification of a normal copy of the gene will give a larger PCR product than the amplification of the mutated gene, which yields a PCR product.

Objective: To test for the presence of the defective CFTR gene using PCR.

Materials (per group):

  • 1 tube containing 60 μL of PCR master mix

  • 1 tube of 20 μL of extraction buffer for each of the patients who had a positive IRT test

  • One 20–200 μL or 10–100 μL micropipette and a box of yellow tips

Procedure:

  1. Collect the appropriate extraction buffer tubes from your instructor.

  2. Add 20 μL of master mix to each tube and label the tube with the patient number.

  3. Add one-quarter of the appropriate paper punch to each tube.

  4. Place the samples in the thermacycler.

You will receive the results of the PCR reaction and electrophoresis during the next class period so that you can analyze the DNA of the patients.

Reference data:Figure 2 shows the expected results for each of the controls.

Figure 2.

Expected results for each of the controls. Lane A: homozygous dominant genotype. Lane B: heterozygous genotype. Lane C: Homozygous recessive genotype.

Figure 2.

Expected results for each of the controls. Lane A: homozygous dominant genotype. Lane B: heterozygous genotype. Lane C: Homozygous recessive genotype.

Data:

SampleInterpretation
Control A  
Control B  
Control C  
Patient 1  
Patient 2  
Patient 3  
Patient 4  
SampleInterpretation
Control A  
Control B  
Control C  
Patient 1  
Patient 2  
Patient 3  
Patient 4  

Analysis and Recommendations:

  1. Explain your interpretation of the results in sentences.

  2. Who should have the sweat test? Why?

Third Screening Test: Sweat Test

Objectives: To see whether the patient expresses the phenotype associated with the gene mutation for CF, and to test for an increase in chloride ion concentration in the sweat of the patient. (Note: Students should have only one patient to test.)

Procedure: Use the conductivity meter to measure the chloride ion concentration in the sweat collected from the patient.

  1. Turn the meter on by pressing the black button on the top.

  2. Place the meter into the sweat sample.

  3. Swirl the beaker containing the sweat sample (DO NOT swirl the meter).

  4. When the reading is stabilized, record the data.

  5. Repeat for each sample that you determined needed testing.

Reference data (CFF, 2013):

PatientChloride (mmol/L)Sodium (mmol/L)Conductivity Test (mmol/L)
Normal CFTR 15.5 17 30–59 
Mutated CFTR 114 107 >60 
PatientChloride (mmol/L)Sodium (mmol/L)Conductivity Test (mmol/L)
Normal CFTR 15.5 17 30–59 
Mutated CFTR 114 107 >60 

Data:

SampleConductivity Test Results (mmol/L)Interpretation
Normal control   
Abnormal control   
Patient no.   
Patient no.   
Patient no.   
Patient no.   
SampleConductivity Test Results (mmol/L)Interpretation
Normal control   
Abnormal control   
Patient no.   
Patient no.   
Patient no.   
Patient no.   

Diagnosis and recommendations:

What is your final diagnosis for each of the patients?

What recommendations would you make to the parents?

End-of-activity questions:

Discuss these questions with your lab group before you leave today, and write your answers in your notebook.

  1. What is the purpose of doing multiple tests?

  2. What is each test looking for?

  3. Why is the sweat test considered a confirmatory test?

  4. Do you think that carriers of CF should be notified? Why or why not?

  5. Do you think that the future spouses of CF carriers should be tested to see if they are carriers as well? Why or why not?

Laboratory report: Write the following letters in your notebook.

  1. Write a letter to the parents of the patients who have been diagnosed with cystic fibrosis. In your letter, explain what the disease is, how it is inherited, what protein is affected, and how it was diagnosed (include an explanation of the tests, the patient’s results, and an explanation of what they mean). Explain the benefits of catching the disorder early and refer the parents to a CF center for additional support.

  2. Write a letter to the parents of any patient who is a carrier of the CF gene. Explain what it means to be a carrier. Should anyone else in the family be tested for the gene? Why or why not? If two people are heterozygous for a CF mutation, what are the chances that their child will have CF?

Instructor’s Notes

There is no actual blood used in this experiment. The red “blood” on the Guthrie cards is just red marker. The “extraction buffers” in the labeled tubes contain the solutions that give the students the various results. This laboratory exercise can be run using actual experiments/kits (A), using simulations of actual experiments (B), or using both. How you to choose to run the exercise will depend on your budget, the equipment available, and the time you have allotted for the exercise. Students should work in lab groups of four. Leaving room for four patients to be listed on all data sheets allows the students to decide which patients need to be tested in each case.

1. ELISA

(A) This experiment can be performed using Bio-Rad’s ELISA Immuno Explorer Kit (or another commercially available ELISA kit). Students will be working with four “patients”; two of these patients will have a positive result, and two will show a negative result. The experiment needs to be set up so that the extraction buffer tubes contain different substances, based on the desired results. The antigen supplied with the kit is placed in two microcentrifuge tubes, and water is placed in the other two tubes. The tubes are numbered 1–4 and labeled as extraction buffer. You can choose which two samples are positive and which are negative. These can be the same for each group (which makes the setup easier), or they can be different for each group (just make sure that each group gets both positive and negative samples). Follow the kit directions for preparing and setting up the other reagents.

(B) If the cost of purchasing the ELISA kit is prohibitive, the ELISA can be simulated by using 10M NaOH in place of the antigen, water can be used for the negative control and antibodies, and phenolphthalein can be used as the substrate/chromogen.

2. PCR Reactions & Gel Electrophoresis

This is the most expensive and time-consuming part of the entire exercise. If students run an actual PCR reaction and gel electrophoresis, you will need to have the equipment available to do so. It will take at least two class periods before they get their results, one to set up and run the PCR reaction and another to run and visualize the gel. Described below are three ways to set up this part of the lab, each with varying time and cost factors. The results should show that one patient carries two recessive alleles and the other patient is heterozygous. (Two of the patients have been eliminated at this point because of normal IRT ELISA results.)

(A) Students perform both the PCR reaction and gel electrophoresis. Since there are no commercially available kits for the screening of the CFTR gene, you can use Bio-Rad’s PV92 PCR Kit refill package. The PV92 PCR kit refill package contains PCR primers, controls, and PCR master mix. Each group of students will receive two PCR microcentrifuge tubes; one will contain the primers and the homozygous control (−/−), and the other will contain the primers and a heterozygous control (+/−). Both of these tubes are labeled as extraction buffer. Students perform the PCR reaction as described. Since each group of students has a different set of patients, it does not matter which paper sample they place into which tube. Since most academic thermocyclers take 2 hours to complete the reaction, students place their tubes in the thermocycler, the reaction is run, and the tubes are then refrigerated until the next lab period. Supply the students with appropriate controls to run on the gel with their samples. The students then run samples on the electrophoresis gel. You can prepare the gels and buffer tanks for the students or have them do it. Be sure to follow the PV92 PCR protocol. Depending on the length of your lab periods, it may take more than one lab period to complete.

Another option is to have the students perform the PCR reaction, and the instructor runs the gels for the students before the next lab period. Using this method, the students run the PCR reaction as set up above, but the instructor runs the electrophoresis gels and performs the staining/destaining procedure so that when the students return to the next lab class, they have gels to analyze. This method requires the careful labeling of student tubes so that each group receives gels with the proper samples on them.

(B) Students use simulated PCR reagents and photos of the gels to analyze. This is the easiest and most cost-efficient method and is recommended for classes that lack the equipment, such as a thermocycler and/or electrophoresis equipment, to perform the experiments. With this simulated method, students receive tubes of water labeled “extraction buffer” and “master mix.” They still gain valuable experience working with micropipettes and an understanding of the techniques and reactions involved in PCR, without using the expensive reagents. Once the students set up the reaction, they turn their tubes into the instructor, who then “sends them off” for processing and gives the students a photo of the results to analyze.

3. Sweat Test

The sweat test is a conductivity test performed using a conductivity meter. It can be set up using the calibration solutions that are recommended for the conductivity meter or using water and salt solutions. You should provide the students with a positive control and a negative control as well as one positive patient sample. If you do not have a conductivity meter, you can just provide the students with data to analyze.

References

References
American Association for Clinical Chemistry. (2012). Lab Tests Online: Trypsinogen. Available at http://labtestsonline.org/understanding/analytes/trypsinogen/tab/glance.
Bowling, F.G. & Brown, A.R.D. (1988). Newborn screening for cystic fibrosis using an enzyme linked immunoabsorbent assay (ELISA) technique. Clinica Chimica Acta, 171, 257–261.
Cystic Fibrosis Foundation. (2010). Testing for Cystic Fibrosis. [Online.] Available at http://www.cff.org/AboutCF/Testing/.
Lap-Chee, T. (1995). The cystic fibrosis transmembrane conductance regulator gene. American Journal of Respiratory and Critical Care Medicine, 151(Supplement), S47–S53.