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II. |
Purpose
of Case It! Investigator and instructions
for use |
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III. |
Purpose
of the Case It! simulation software and instructions for use |
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Example cases |
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A. Human genetic diseases |
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B. Infectious diseases |
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1. HIV/AIDS |
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C. Forensics |
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D. Phylogenetic studies |
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1. Primate
relationships - human, chimp, gorilla |
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E. Simulation of wet labs |
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2. Mapping
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VI |
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Developed
by Karen Klyczek, Kim Mogen, Douglas Johnson, Mary Lundeberg, and Mark Bergland,
University of Wisconsin-River Falls, with contributions from UWRF Biology
students and a network of beta-testers in the U.S. and Canada. Contact mark.s.bergland@uwrf.edu for additional
information. This project was supported,
in part, by the National Science Foundation. Opinions expressed are those of the authors
and not necessarily those of the Foundation.
Copyright 2006
.
Case It! is NSF-supported project initiated in
1995 by participants in a BioQUEST Curriculum Consortium workshop. The overall
goal is to develop a framework for collaborative case-based learning in molecular
biology using interactive computer simulations, and to have students from
around the world participate in web-based “poster sessions” via Internet conferencing.
An automated system allows students to create posters by entering text
and uploading graphics to a server located at UW-River Falls; no knowledge
of html is required. The system is integrated with an electronic
bulletin board so that collaborative teams of students can discuss their results
with students at other institutions.
Software modules described in this manual can be
downloaded from the Case It web site at no cost to educators (http://www.uwrf.edu/caseit/caseit.html). Results of class-testing are also available
at this web site. Contact mark.s.bergland@uwrf.edu) for additional
information concerning the project, and to learn how you and your students
can participate.
Purpose: Case It!
Investigator is a software tool that students can use to gather background
information on cases using internal links within Investigator itself, and external links
to Internet sites. In essence, Investigator is an interactive version
of this Resource Manual with hyperlinks to various sections of the manual,
viewed as html pages (a password is required for access to the Key to Cases
section, however. Instructors can obtain
this password by emailing mark.s.bergland@uwrf.edu). In addition, Investigator includes pop-up menus
which enable users to access important Internet sites providing background
information. Version 2.0 of Investigator includes video clips for
selected cases.
Instructions
for use: Click the Start button on the opening screen,
and the main screen for Investigator
will appear. Click the question mark
icon to activate instructions, which will appear as the cursor is rolled over
various regions of the screen. Click
the question mark a second time to deactivate the instructions.
Any words in blue are hot links either to internal
pages or Internet sites. To navigate
backwards and forwards, click either the blue words in the outline at upper-left,
or the arrow buttons on the button bar. Web sites visited via buttons on the button
bar will be listed in the window at upper-right; you can revisit these sites
by clicking any of the listings in this window.
Clicking the “Save” button will allow you to save any Investigator page as a text file. Pages can be printed by clicking on the "Print"
button in the Windows version; Mac users must first save files, and then they
can be opened in Word and printed. If you get an error message when you click the
Print or Save buttons, open the file of interest directly in your browser,
then print or save it (the files are located in the “html files” folder inside
the “Case It Investigator” folder).
The “Tools for Case Analysis” button enables users
to start any other software application, assuming that the application itself
(or a shortcut to the application) is inside the “Case It Investigator” folder.
By default, the pop-up menu associated with this button lists “Shortcut
to Case It! Version 5.0 beta.exe (Windows) or CaseItv5.0beta alias (Mac)".
The "Resources(1)" and "Resources(2)" buttons will
open up your browser to specific web pages.
You can modify these and other pop-up menus to include other applications
and web pages, and you also can replace or edit the internal html pages if
you so desire. Instructions for doing
so can be found by clicking the “How to customize Case It! Investigator” link
on the main screen of the program.
Case It! is an open-ended simulation which includes
methods for analyzing DNA (restriction digestion and mapping, polymerase chain
reaction (PCR), DNA electrophoresis, Southern blotting and dot blotting analysis)
and proteins (protein electrophoresis, Western blotting, ELISA).
The restriction digestion simulation will cut any DNA sequence with
any combination of restriction enzymes and runs realistic gels and Southern
blots of the resulting DNA fragments. Eight
wells are available for loading, with up to 40 fragments per well visible
on the screen. The simulation cannot separate fragments larger
than 38 kb on a 1.2% agarose gel, or larger than 94.2 kb on a 0.4% agarose
gel. This is not a difficulty for any
of the sample cases included with the Resource Manual, as fragment sizes are
smaller than these maxima. The size
of any large fragment can be shown, however, using the Gel menu, which is
accessible from the Gel/Blot or Lab Bench screen. Protein electrophoresis and Western blots can
also be run on the Gel/Blot screen. Acrylamide
concentration of 5-18% are available for protein gels.
The PCR simulation will generate the DNA fragments
that would be amplified using the primer sequences supplied. These fragments can be run in the gel electrophoresis
simulation to analyze the sizes of the fragments or to carry out Southern
blotting, or they can be used in the dot blot simulation. PCR also can be run in a 96-well plate, for
cases where PCR is used to detect or quantify genetic material, e.g. from
a pathogen (NOTE: The results of the 96-well PCR cannot be run on a gel, nor
can they be used as probes or DNA samples in the dot blot). The dot blot simulation will determine whether
there is a match (hybridization) between the DNA applied to a dot and the
DNA added to the hybridization solution. The
dot blot is carried out using a 96-well plate format. The DNA loaded in the well would be labeled
and would hybridize to the dot DNA if there is a sequence match. Hybridization is detected by the dot turning
dark as a result of the labeled DNA sticking to it. The relationship between a probe and a DNA sample
may be the reverse of what is used for Southern blotting, in that unlabeled
probe is often applied to the dot, while the sample DNA (e.g. from a patient
in a human genetic disease case) is labeled (usually by PCR amplification
using labeled primers) and allowed to hybridize to the probe.
The ELISA simulation can be set up to detect either
antibodies or proteins. If proteins
are being detected, the well of the ELISA plate are coated with antibodies
and protein samples are added. Some
cases require detection of antibodies in samples, so the wells are coated
with protein. A positive ELISA result
will produce color for which absorbance values can be reported.
There are five main screens in the Case It! simulation:
DATA SCREEN, GEL/BLOT SCREEN, ELISA/DOT BLOT/96-WELL PCR SCREEN, GENBANK
SCREEN, and LAB BENCH. In addition,
there is a PHOTO SCREEN for documenting gel results.
Brief instructions for each screen are given here to provide an overview
of the program; they also appear in the program’s Help window.
An extensive online tutorial is available from the Case It! home page
and also via the "Tutorial" menu in the program.
Use the DATA
SCREEN to
quickly digest DNA and load it into wells, to generate PCR fragments and load
them into gels, or to load proteins and antibodies for a Western blot, if
appropriate.
To digest
DNA and load gels:
1.
Use the DNA menu
to select a DNA sample. There is now
no limit to the size of the DNA file that can be viewed via the "open
and show DNA" menu selection.
Note: Each
case has the necessary DNA files packaged into a folder. These individual case folders can be found the
Cases folder within the main Case It! folder/directory. Multiple files can be opened at once by pressing
the Shift key while selecting files.
a. A list of opened files appears in the Opened/Processed
window; clicking on a file makes it the active file, which appears in the
main window of the data screen,
b. The lower window of the data screen shows
the number of individual fragments in the file, and the size or sequence of
each. Click on the check box for Size
or Sequence, and click on the number of the fragment you want to view. The order of the fragments can be changed by
using the Options button.
2. Use the SITE menu to open an enzyme file.
Alternatively, enzymes can be selected by clicking the "enz"
button, which activates a pop-up menu showing graphical
depictions of enzyme cut sites.
3. Use the DIGEST menu to digest the DNA.
4. Click a well button, then click the 'load'
button.
5. Repeat steps 1-4 to digest and load additional
samples.
6. Use the RUN menu to run the gel. Note that the default setting results in bands
that are barely visible in the gel. Use
the color buttons below the gel to select color options that simulate Ethidium
bromide (fluorescent orange) or methylene blue staining, or an ultraviolet
light photo of Ethidium bromide staining (white bands on dark background).
7. If running a Southern blot, select a probe
file using the SITE menu.
8. Use the RUN menu to run a Southern blot.
9. To copy and paste from Loaded DNA window to
Gel Label:
-Select text you wish to copy in the Loaded DNA window.
-Click the COPY button in the Loaded DNA window.
-Click PASTE in the Loaded DNA window to paste copied text into
the Gel Label.
10. Use the EDIT menu to clear and reset all parameters
NOTE: If the 'show genomic smear' option is selected
in the 'Genomic' menu, a smear will cover the actual DNA file fragments whenever
a DNA file ending with the suffix '.gen' is loaded and run.
Using the slider feature, the smear can be made more or less opaque
to cover or reveal the fragments. A Southern blot can then be run to reveal the
fragments of interest. Note also that
the genomic smear does not affect migration values and fragment sizes shown
in the 40 boxes to the right of the gel; these values are generated from digestion
of the DNA file sequences.
To run the PCR procedure:
1.
Use the DNA menu or button to
select a DNA sample.
2.
Use the SITE menu to select a
single file containing the forward and reverse PCR primers.
3.
Use the RUN menu and select “Run
PCR”.
Options and notes:
To show the double-stranded
configuration for the active DNA sequence, select “Show DNA base pairs and
primers” form the DNA menu. This option
enables you to find forward and reverse primer sites on the sequence.
You can load the resulting
PCR products (the fragment could also be digested before loading, if appropriate)
or load it into a well on the ELISA/Dot Blot. You also can use the “Run” menu to show the
PCR product or save it as a text file.
DNA can also be loaded into
the gel or dot blot via the “Opened DNA” window. Clicking once on a DNA sample highlights this
sample and allows it to be loaded. Click
on the question mark in the Opened DNA window for more information.
4. Alternatively,
the DNA and primers can be loaded into a 96-well plate. (NOTE: The 96-well PCR is for quantitative PCR applications
only, e.g. viral load determination. The PCR product from the 96-well reaction cannot
be run on a gel, nor can it be used as a probe or as a DNA sample in a dot
blot.)
a. Go to the ELISA/Dot Blot/96-well PCR screen.
Click on the Method button and selected PCR 96-well
b.
Use the Open button to open DNA and
primer files
c. Select
wells by clicking and dragging around the well(s), then click the Load button.
The loaded samples will be listed automatically on the spreadsheet
next to the plate.
d. Click the Run button to run the PCR. A fluorescent signal will appear if there is
a positive reaction. If appropriate
for the case, use the Option button to report
Viral load values. A photo can be saved
by clicking on the Option button and selecting Photo.
Gels can also be set up and run on the LAB BENCH using simulated laboratory equipment:
1. Use the TOOLS menu to assemble a gel box
(or to get a preassembled gel box).
2. Use the DNA menu to select up to two DNA
samples.
3. Use the SITE menu to select up to four enzymes.
4. Add enzymes to the DNA tubes using the micropipette.
5. Use the TOOLS menu to get a Heat Block.
6. Drag the DNA tubes into the Heat Block.
7. Click the Heat Block switch (top view) to
digest the DNA.
8. Drag the DNA tubes from the Heat Block.
9. Use the micropipette to load wells in the
gel box.
10. Use the TOOLS menu to get a Power Source.
11. Run the gel by turning on the Power Source.
12. Use the SITE menu to select a probe.
13. Use the RUN menu to run a Southern blot.
14. Use the EDIT menu to clear and reset all parameters
before running another gel.
The GEL PHOTO SCREEN is used to compare photos
taken of current gels or blots and also to view photos taken of previous gels
which have been saved as JPEG files. Labels
are editable, and information can be copied and pasted from the Well Data
window to any photo label.
1. Use the SAVE buttons to save photo 1 or photo
2 as JPEG files.
2. Use the REPLACE buttons to replace the photos
on this screen with previously saved gel photos.
3.
Use the ROTATE buttons to rotate
horizontal photos into a vertical configuration (PC version only).
Use the ELISA/DOT BLOT SCREEN to determine whether there
is a sequence match between DNA samples and probes.
·
Use the Method
button to select Dot blot and to indicate whether DNA samples or probes will
be spotted.
·
Open DNA and
probe files, and select wells by clicking and dragging. Click the Load button to load the well.
·
Click the Run
button. Positive dots will turn dark,
if there is a sequence match.
·
Use the Options
button to save a photo of the results.
Note: Labels will be filled in automatically with
file names associated with particular wells; these labels can be edited before
the photo is taken.
To run a protein gel or
a Western blot on the DATA SCREEN
1. Use the Protein menu or button to open a protein
file. As with DNA files, opened files
appear in the Opened/Processed window, and sequences or sizes can be viewed
in the lower Data Screen window.
2. Click or drag wells to load them.
3. Click the Run button to run the protein gel.
4. To run a Western blot, use the Antibody button
or Site menu to open antibody files. Clicking
the number next to a well associates the open antibody with that well lane.
5. Click the Run button to run the Western blot.
Use
the ELISA/DOT BLOT screen to detect
the presence of specific antibodies or proteins in a sample.
1.
Click on the Method button to select
ELISA and to indicate whether the samples are antibodies or proteins.
2. Use the Open button to open protein and antibody
files.
3. Click and drag around wells to select them.
Then click Load to load them with the active file.
The labels will automatically be filled in.
4. Click the Run button to run the ELISA. Blue color will appear in wells where there
is a match between the antibody and protein.
Use the Option menu to record Absorbance or Transmittance data.
5. A photo can also be taken using the Options
button.
The GENBANK
SCREEN
enables you to access the GenBank web site and filter sequence files so that
they are ready for analysis:
1. Use the GENBANK menu to automatically open
your web browser to the GenBank web site.
2. Save any part of the GenBank sequence as a
text-only file.
3. Use the FILE menu to open the saved GenBank
sequence.
4. Use the FILTER menu to remove everything but
the DNA sequence from the GenBank file.
5. Digest the DNA sequence using either the Data
Screen or the Lab Bench.
IV. Suggestions for Class Use of Case It!
The example cases described
here were developed for use in introductory undergraduate biology classes
to help students deal with concepts and issues in molecular biology, but they
can be adapted to a variety of educational settings. Some of the approaches that can be employed
when using these examples in classes are described below.
Each case description includes
the case scenario and instructions for analyzing the case, as well as background
information and discussion questions. The cases can be presented to students using
this format, having them read the background information and perhaps do some
additional research, then carry out the analysis, interpret the results and
discuss the significance and the issues raised.
Alternatively, instructors can edit the cases to add or omit information
as appropriate for the backgrounds of students and the course objectives. Students may be required to:
• focus on the ethical and social issues raised by the analysis
and the decision-making process involved.
• take on a particular role, e.g. genetic counselor or family member,
and present the case interpretation from that
perspective.
• develop hypotheses about the results, based on the background
information about the molecular biology in the case, before running the analysis
• start with the case analysis and results, and carry out their
own research to obtain information necessary to interpret the case.
In addition to using these
cases and sequences, the module allows instructors to develop their own cases
using DNA sequences obtained from GenBank or elsewhere (see "Building
your own case study" on page XX). Sequences, restriction
enzyme sites, probes, primers and antibodies all are editable text files. Case development also can be assigned to students
in more advanced biology courses. The
student-designed cases then can be subjected to peer review via poster presentations,
etc. and used by students in introductory courses.
V.
Example cases
The DNA and protein sequences
for the cases described here are located in the Cases folder that is downloaded
with the Case It! Software simulation. The necessary enzymes, probes, antibodies, or
proteins for a particular case will be located in the same folder as the DNA
sequences. Keys to the cases are found
beginning on page XX
A. Human genetic diseases
Genetic diseases are caused by alterations in the DNA which result in loss of function or altered function of a protein. These changes in the DNA can be detected, even in the absence of disease symptoms, by isolating DNA from the patient and using restriction enzyme digestion and Southern blotting.