Case-based Learning Using Protein and DNA Simulations to Analyze Cases Based on Infectious

DiseasesMark Bergland and Karen Klyczek, University of Wisconsin-River FallsMark Bergland and Karen Klyczek, University of Wisconsin-River Falls

OverviewOverview

•Description of Case It! ProjectDescription of Case It! Project• Sample case study in human genetic diseaseSample case study in human genetic disease

•Hands-on demonstration of DNA analysis Hands-on demonstration of DNA analysis softwaresoftware

• Bacterial DNA analysis (new!)Bacterial DNA analysis (new!)

•Hands-on demonstration of protein moduleHands-on demonstration of protein module• Sample HIV case studySample HIV case study

• Other infectious disease case studies (if Other infectious disease case studies (if

time permits) time permits)

Case It! ProjectCase It! Project

URL for URL for Case It! Home Page:Case It! Home Page:http://caseit.uwrf.edu (please (please register)register)

Contact: mark.s.bergland@uwrf.eduContact: mark.s.bergland@uwrf.edu

Supported by the CCLI program of Supported by the CCLI program of the the National Science National Science FoundationFoundation

Case It! ProjectCase It! Project

Electronic framework for analyzing Electronic framework for analyzing and discussing case studies in and discussing case studies in molecular biologymolecular biology

•Genetic and infectious diseases and Genetic and infectious diseases and associated ethical associated ethical issuesissues

•Students gather background information Students gather background information on cases on cases

•Analyze DNA and/or protein sequences Analyze DNA and/or protein sequences using using Case It! Version 5.02 betaCase It! Version 5.02 beta

•Prepare web-page poster using automated Prepare web-page poster using automated systemsystem

•Poster session via Internet conferencing Poster session via Internet conferencing

•Role-playingRole-playing

Techniques for DNA Techniques for DNA analysisanalysis

Features of Case It! Version 5.0Features of Case It! Version 5.0

• DNA and protein electrophoresisDNA and protein electrophoresis• Restriction enzyme digestion and mapping Restriction enzyme digestion and mapping • Southern blottingSouthern blotting• Dot blotting Dot blotting • Polymerase Chain Reaction (single and 96-Polymerase Chain Reaction (single and 96-well)well)

• ELISAELISA• Western blottingWestern blotting

Case It! web editorCase It! web editor

Features of the Case It! web Features of the Case It! web editoreditor

• Web page constructionWeb page construction• Conferencing/discussion board Conferencing/discussion board • Collaborate with classes at other Collaborate with classes at other institutionsinstitutions

• Instructors can add classes Instructors can add classes • Upload class roster, organize teamsUpload class roster, organize teams

Sample case: Huntington’s Sample case: Huntington’s DiseaseDisease

Susan is a 23-year-old whose father, age 55, and Susan is a 23-year-old whose father, age 55, and paternal aunt, age 61, have been diagnosed with paternal aunt, age 61, have been diagnosed with Huntington’s chorea. A paternal uncle, age 66, Huntington’s chorea. A paternal uncle, age 66, appears to be unaffected by the disease. Susan appears to be unaffected by the disease. Susan wants to know if she inherited the mutated gene wants to know if she inherited the mutated gene from her father so that she can prepare for that from her father so that she can prepare for that future if necessary. She arranges to undergo DNA future if necessary. She arranges to undergo DNA testing for Huntington’s. Her 17-year old testing for Huntington’s. Her 17-year old brother, John, also decides to be tested after brother, John, also decides to be tested after talking with Susan.talking with Susan.

Ethical issues raised by Ethical issues raised by the casethe case

Should someone with a family history of HD Should someone with a family history of HD be required to undergo testing? Should be required to undergo testing? Should they have children if they test positive, they have children if they test positive, or if they have not been tested?or if they have not been tested?

Should someone as young as John be tested?Should someone as young as John be tested?

Should the results of such tests be made Should the results of such tests be made available to insurance companies? to available to insurance companies? to potential employers? to potential mates?potential employers? to potential mates?

Case analysisCase analysis

Case It! InvestigatorCase It! Investigator is used to gather is used to gather background information on casebackground information on case

Case It! Version 5.0 betaCase It! Version 5.0 beta is used to is used to analyze DNA sequences associated with caseanalyze DNA sequences associated with case

Case It! Launch PadCase It! Launch Pad gives access to a web gives access to a web editor/conferencing system editor/conferencing system

--electronic poster sessionselectronic poster sessions-role playing-role playing

Online tutorialOnline tutorial gives detailed gives detailed instructions instructions (e.g. S. blot)(e.g. S. blot)

Bacterial DNA analysisBacterial DNA analysis

Analysis of total genomic DNAAnalysis of total genomic DNA» Restriction enzyme digestion (pulsed-Restriction enzyme digestion (pulsed-field gel electrophoresis (PFGE))field gel electrophoresis (PFGE))

» Southern blotSouthern blot» PCRPCR

Example: SalmonellaExample: Salmonella» Food poisoningFood poisoning» Zoonotic transmission from pets Zoonotic transmission from pets (turtles, rodents)(turtles, rodents)

PFGE to identify Salmonella strainsPFGE to identify Salmonella strains(Nagano et al., Jpn. J. Infect. Disease 59:132, (Nagano et al., Jpn. J. Infect. Disease 59:132,

2006) 2006)

Using PFGE to generate treesUsing PFGE to generate trees

((Woo and Lee, J. Microbiology, Feb 2006, p. 106)Woo and Lee, J. Microbiology, Feb 2006, p. 106)

Multiplex PCR to distinguish Salmonella strains(Alvarez et al., J. Clin. Microbiology, Apr 2004, p. 1734)

Run Case it simulation (v5.03 beta) to demonstrate these techinques – restriction enzyme digestion, DNA electrophoresis, Southern blot, and PCR

Case It simulation runs gels horizontally, so previous two photos have been rotated for comparison…

Note: V5.02 currently available for downloading

Using Case It to simulate Salmonella studies

Protein moduleProtein moduleCase It! Version 5.0Case It! Version 5.0

• ELISA (Enzyme immunoassay)ELISA (Enzyme immunoassay)

• Protein gel electrophoresis/ Western Protein gel electrophoresis/ Western blotblot

• Infectious disease cases Infectious disease cases » HIVHIV» SARSSARS» West Nile virusWest Nile virus» InfluenzaInfluenza» EbolaEbola

ELISA test results

HIV viral proteinsHIV viral proteins

gp16gp1600

Precursor of Precursor of envelope protein envelope protein (env)(env)

gp12gp1200

Outer envelope Outer envelope proteinprotein

(env)(env)

p55p55 Precursor of core Precursor of core proteins (gag)proteins (gag)

p41p41 Transmembrane Transmembrane envelope protein envelope protein (env)(env)

p32p32 Integrase (pol)Integrase (pol)

p24p24 Inner core protein Inner core protein (gag)(gag)

Western BlotWestern BlotBand pattern interpretationBand pattern interpretation

Lane 1, HIV+ serum (positive control) Lane 2, HIV- serum (negative control) Lane A, Patient A Lane B, Patient B Lane C, Patient C

No bandsNo bands NegativeNegative

Bands for two of Bands for two of these three these three proteins: gp120, proteins: gp120, gp41, p24gp41, p24

PositivePositive

Any other band Any other band patternpattern

IndeterminIndeterminateate

Band interpretation:

from www.biology.arizona.edu

HIV Case studiesHIV Case studies Case scenario - Case scenario - videovideo and text (HIV – and text (HIV – Anna), using Anna), using Investigator v2Investigator v2

ELISAELISA test - initial screening test - initial screening

Western blotWestern blot to follow up ELISA results to follow up ELISA results

PCRPCR to measure viral load to measure viral load

Upload graphical results to Upload graphical results to Case It web Case It web editoreditor

Case it Web EditorCase it Web Editor

URL: URL: https://caseit.uwrf.eduhttps://caseit.uwrf.edu

Username: Username: guest.student@class.siteguest.student@class.site

Password: hidFavgiPassword: hidFavgi

Comments from Zimbabwe Comments from Zimbabwe studentsstudents

“To me it was an eye opener… Since Zimbabwe is hard hit by HIV it is important to have this project CASE IT since it affects the students’ lives at large. The videos shown are touching. These can help students feel it.”

“It was also interesting to network with international students via conferencing. Indeed, the world is a global village.”

“(I) was able to get an understanding of how American families react to their family members when they have tested HIV positive”

Comments from Zimbabwe Comments from Zimbabwe studentsstudents

“I had no idea how I could do practicals on H.I.V testing given the limited apparatus and financial limitations in my school.”

“I found the use of the computer very useful in simulations because it enables the carrying out of experiments which would otherwise be impossible due to the costs involved”

Comments from Zimbabwe Comments from Zimbabwe studentsstudents

“The programme was also very interesting because it had cases of real people that got infected in different ways and situations … and the counselling they were given and how some observed the advice and some ignored this advice got themselves into worse trouble.”

Comments from Zimbabwe Comments from Zimbabwe studentsstudents

“The subject still remains every teacher’s concern. Of particular concern however, is the increase in the number of orphans. It is estimated that by the year 2005 approximately 910000 children will have lost their parents to AIDS. Poverty, depression, and stigma will haunt most of these children. They will most likely expose themselves to HIV infections in pursuit of basic need and will painfully erode their basic right to family, protection health and food. Our challenge is therefore to intensify awareness education to these children.”

Invitation to Invitation to participateparticipate

• We welcome participation in the We welcome participation in the Case It! ProjectCase It! Project

• No cost to educatorsNo cost to educators• High school and university High school and university educators are invitededucators are invited

• Contact mark.s.bergland@uwrf.eduContact mark.s.bergland@uwrf.edu