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Simulated Search For A Disease Gene

By Katharine Noonan



Type of Activity:

  • Hands-on activity
  • Simulation
  • Group/
    cooperative learning

Target Audience:

  • Advanced/AP Biology
  • Anatomy And Physiology
  • Genetics
  • Biotechnology

Background Information:

Teacher notes:

Reserve use of electrophoresis equipment and order DNA and enzymes to arrive before lab. Plasmid 1 and Plasmid 2 should be the same size, one with one restriction site for the restriction enzyme, the other with 2 restriction sites for that enzyme.

The plasmids I used are not commercially available. Two plasmids available from Sigma should work: pBR322 for Plasmid 1 (product number D4904) and FX174 for Plasmid 2 (product number D5401). pBR322 has 1 site for the enzyme Nru1 and FX174 has 2 sites for Nru1 1. Nru1 is also available from Sigma (product number R5759. The enzyme comes with about 1.5 ml of reaction buffer which can be used for dilution according to directions. If you have questions, call the Sigma technical help 800 number. They are very nice. ). Lambda DNA and HindIII are also available from Sigma.

Store DNA and enzymes as directed in freezer or refrigerator:

DNA:

  • 120 ml of Lambda DNA stock (stock is 0.1 mg/ml) -- (8 ml/ group)
  • 240 ml of plasmid 1 DNA stock (stock is 0.1 mg/ml)
  • 96 ml of plasmid 2 DNA stock (stock is 0.1 mg/ml)
Enzymes:
  • 130 ml Nru1 stock (stock is 10-20 units/ml) Dilute with 425 ml reaction buffer within 1/2 hour of lab -- (37 ml of diluted enzyme/group).
  • 26 ml HindIII stock (stock is 10-20 units/ml). Dilute with 85 ml reaction buffer within 1/2 hour of lab -- (6 ml of diluted enzyme/group).

Label microfuge tubes with permanent marker. Aliquot DNA any time before lab. Dilute and aliquot enzymes just before the lab. Label and aliquot:

  • 30 tubes of Lambda DNA -- aliquot 8 ml/tube of Lambda stock.
  • 15 tubes of HindIII -- aliquot 6 ml/tube of diluted HindIII stock (see above).
  • 15 tubes of Nru1 -- aliquot 37 ml/tube of diluted Nru1 stock (see above).
  • 45 tubes of simulated human DNA -- aliquot 4 ml/tube of Plasmid 1 DNA (homozygotes -number the tubes in triplicate with the following patient numbers (see pedigree): 11, 14-16,23, 26-28, 31,36,38,47-49, 52.
  • 30 tubes of simulated human DNA -- aliquot 2 ml/tube each Plasmid 1 and Plasmid 2 DNA (heterozygotes) -number the tubes in triplicate with the following patient numbers: 13, 21, 22, 24, 25, 32, 33, 34, 35, 45
  • 9 tubes of simulated human DNA -- aliquot 4 ml/tube of Plasmid 2 DNA (homozygotes). -number the tubes in triplicate with the following patient numbers: 12, 44, 51

Background required of students:

Students should be familiar with Mendelian genetics, pedigrees and their interpretation, DNA structure and function. Students should understand restriction analysis and RFLP use. I assigned pages 148 to 155 in Micklos and Freyer (1991). Experience with electrophoresis and use of micropipettors is helpful. Videos showing Wexler's pedigree wall and fieldwork in Venezuela are useful. Details of probes, blotting techniques, cloning, chromosome walking, and reverse genetics may be explored with advanced students.

Preparation time needed: 2-6 hours

Class time needed:

4 to 8 days total: one to three periods to set the stage and review background concepts; one period for digest, one period for electrophoresis, 1-3 days for discussion and follow-up activities.


Project:

Abstract

This simulation is based on the research of Nancy Wexler and James Gusella on Huntington's disease (see Micklos and Freyer, 1991, DNA Science, pp. 148-155). Plasmid DNA is used to represent human DNA samples from a family affected by a genetic disease. RFLP analysis of the samples reveals a potential marker for the disease gene. A mutation within or near the disease gene has created a new restriction site for the restriction endonuclease Nru1, yielding 2 smaller restriction fragments on electrophoresis instead of a single larger one. The students discover that the disease phenotype is linked to the double-banded allele. They are able to use the information to describe the inheritance of the disease (autosomal, recessive) and to predict that a fetus (#52) will be unaffected by the disease. Through creative writing assignments, students explore personal and societal issues surrounding genetic testing.

Materials needed for 15 lab groups:

For restriction digest:

  • 37 degree water bath; microcentrifuge (helpful, but not essential)
For electrophoresis:
  • 15 gel boxes with power supply
  • 15 gel beds with combs
  • 15 micropipettors (2-10 ml)
  • Tips
  • Larger micropipettor for teacher use (10-100 ml)
  • 15 weighboats for staining
  • Transilluminator
  • Camera and film for photographing gels
  • Spatula and latex gloves for handling gels during staining
  • Goggles

Supplies:

  • Agarose for pouring gels
  • Running buffer to fill gel boxes
  • Loading dye
  • Permanent markers and masking tape
  • 175 microfuge tubes
  • Ethidium bromide stain
  • DNA and enzymes 2X Restriction Buffer for diluting enzymes and DNA
  • Each lab team should have in its rack:
    • 1 tube each: Lambda DNA, Nru1, HindIII
    • 6 tubes patient DNA (randomize numbers and replicates among teams)
    • 1 empty tube for Lambda/HindIII digest (size markers)
  • Butcher paper wall diagram of Jones family pedigree- 81/2" X 11" copy for each student
    Note: Print this document--the numbers in the pedigree will be easier to read.

The Jones Family Pedigree

  • Square indicates a male.
  • Circle indicates a female.
  • Triangle indicates an individual whose sex is not known.
  • "X" indicates a dead individual.
  • Shading indicates individuals affected by the genetic disease (disease phenotype).


Procedure:

  • Day 1: Introduce class to the Jones family. Healthy couple John and Nancy have had a stillborn child. The baby's karyotype is normal and it is not deformed. There is no evidence of infection, but blood analysis shows the accumulation of a lethal metabolite. This chemical imbalance, doctor explains, runs in families. It may kill before or after birth. The location of the faulty gene is not known, nor its protein product. John and Nancy want to know if their second unborn child (#52) will be affected. Discuss evidence from Jones family pedigree. Review pedigrees, Mendelian genetics, linkage.

  • Day 2: Jones family doctor puts them in touch with a research team (the class!) which will examine their DNA for variations (RFLP's) which may be linked to the disease. A closely linked RFLP may allow them to tell whether fetus #52 has the disease. The family's participation in the research could help to locate the disease gene and lead to new diagnostic techniques and treatments (through reverse genetics).

  • Day 3: Students digest patient DNA with Nru1, following modified protocol of Micklos and Freyer (1991) for Lambda digest. They digest Lambda with HindIII to provide size markers. Hold digests over night in refrigerator.

  • Day 4: Subject digests to electrophoresis. Teacher stains and photographs after school.

  • Day 5: Analyze gels. Write genotypes for RFLP's on "pedigree wall" for all family members. Discuss and resolve discrepant findings. Discuss apparent linkage of disease to double-banded marker. Advise Jones family of health of fetus #52. Evaluation: Students take a traditional test about techniques, pedigrees, RFLP analysis, etc., and/or draft a letter to the Jones family reporting and explaining clearly the results of the test. Extension: Through an appropriate piece (letter, diary, advertisement) students explore interests and issues of different people in our society: parents, genetic counselor, family doctor, director of a biotech company interested in marketing diagnostics, insurance company, child #52 when s/he grows up, distant relative, etc. Share aloud with class. Publish. FX174 .Assign a creative writing project in which students take roles of different people in society concerning this scenario.


Acknowledgments:

This project was developed with scientific advice and plasmids developed by Dr. Brian Kearney. Errors in the activity are mine. Mary Connolly and the Lawrence Hall of Science STEPS program also provided support and advice.

1pBR322 is actually about 1kb smaller than FX174, so this pair of plasmids is not ideal. pBR322 should give one 4,361 bp fragment after digestion with Nru1. FX174 will give 2 fragments 2,200 bp and 3,100 bp, approximately. The results should be adequate to simulate an RFLP for this activity.


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