Clinical Laboratory Technology

INTERNET LESSON PLAN NUMBER 3

UNIT - Molecular Techniques

SCOPE OF UNIT:

Top Objectives Overview Materials Procedures Extra Sites Questions Activities Evaluation

TITLE OF LESSON: Tools of DNA Analysis

LESSON OBJECTIVE: The student should be able to:

1. Describe the tools used to analyze DNA
   
   
2. Design a PCR procedure which detects and verifies the presence of a specific gene.
   
   
3. Relate information about source organism, action, and supplier for restriction enzymes.

OVERVIEW:

MATERIALS:

• Computer with Internet access
• Netscape Browser with Shockwave plug-in

PROCEDURES:

Top Objectives Overview Materials Procedures Extra Sites Questions Activities Evaluation

1. Read about the Language of Recombinant DNA.
   
   Access Excellence
   
   URL: http://www.accessexcellence.org/AB/IE/Speaking_Language_rDNA.html
   
   
2. View an overview of DNA manipulation.
   
   Access Excellence
   
   URL: http://www.accessexcellence.org/AE/AEPC/WWC/1993/overview.html
   
   
3. Learn more about restriction enzymes by reading the article and viewing images at these sites.
   
   

   Access Excellence

   Restriction Enzymes
   URL: http://www.accessexcellence.org/AE/AEC/CC/restriction.html
   Restriction Enzyme Action of EcoRI
   URL: http://www.accessexcellence.org/AB/GG/restriction.html
   Cutting and Splicing DNA
   URL: http://www.accessexcellence.org/AE/AEC/CC/plasmid.html
   Examples of Restriction Enzymes
   URL: http://www.accessexcellence.org/AE/AEC/CC/re_chart.html

4. The Polymerase Chain Reaction is described at these sites.
   
   Access Excellence: Polymerase Chain Reaction-Xeroxing DNA
   
   URL: http://www.accessexcellence.org/AB/IE/PCR_Xeroxing_DNA.html
   
   Access Excellence: Polymerase Chain Reaction
   
   URL: http://www.accessexcellence.org/AB/GG/polymerase.html

5. Learn more about recombination at these sites:
   
   

   Access Excellence
   Recombination Up Close
   URL: http://www.accessexcellence.org/AB/BC/Recombination_Up_Close.html
   Inserting a DNA Sample into a Plasmid
   URL http://www.accessexcellence.org/AB/GG/inserting.html
   Cloning into a Plasmid
   URL http://www.accessexcellence.org/AB/GG/plasmid.html
   Transfer and Cloning of the Insulin Gene
   URL http://www.accessexcellence.org/AB/GG/transfer_and.html
   Primer on Molecular Genetics
   Constructing Clones for Sequencing
   URL: http://www.ornl.gov/hgmis/publicat/primer/fig11.html

6. Read the section on Sequence Technologies in the Primer on Molecular Genetics.
   
   URL: http://www.ornl.gov/sci/techresources/Human_Genome/publicat/primer/index.shtml

7. See animations of PCR, Southern Blots, and Cycle Sequencing at the Biology Animation Library.
   
   URL: http://www.dnalc.org/ddnalc/resources/animations.html

8. Identify an unknown bacterium from the base-pair sequence using ribosomal DNA, at the Virtual Bacterial ID Lab.
   
   URL: http://www.hhmi.org/biointeractive/vlabs/

Top Objectives Overview Materials Procedures Extra Sites Questions Activities Evaluation

EXTRA SITES OF INTEREST

• Access Excellence: Monoclonal Antibody Technology-The Basics
  
  URL: http://www.accessexcellence.org/AB/IE/Monoclonal_Antibody.html
  
  
• Monoclonal Antibody Production
  
  URL: http://www.accessexcellence.org/AB/GG/monoclonal.html

URL: http://www.accessexcellence.org/AB/BA/

URL: http://www.ornl.gov/TechResources/Human_Genome/

INTERACTION ITEMS:

1. Once a target sequence is amplified, how will you know whether it’s present?

CLASSROOM, LABORATORY, OR OTHER ACTIVITIES

Top Objectives Overview Materials Procedures Extra Sites Questions Activities Evaluation

Each student will design a PCR procedure to detect an antibiotic resistance gene.

Situation: You are a Laboratory Professional in a public health laboratory. You receive a culture of Staph. aureus which has shown a reduced susceptibility to Vancomycin. Your task is to design a PCR procedure which will detect the vanA gene, if present.

1. Use the European Bioinformatics Institute site to search and retrieve the nucleic acid sequence for the Enterococcus vanA gene.
   • Locate and click the box next to EMBL:EFPVANAG.
   • Under, Perform operation (on the left), selct - on selected.
   • Select FastaSeqs from the view drop down menu.
   • Click the View button
2. View and copy the DNA sequence.
   
   URL: http://www.ebi.ac.uk/
   
   
3. Open a second browser and go to the Primer 3 site to choose primers.
   
   URL: http://frodo.wi.mit.edu
   
   Place the cursor in the box. Paste the sequence into the box and click "pick primers."
   
   *Print out the results.*
   
   
4. To generate a restriction map of the target area, go to the following address:
   
   URL: http://www.firstmarket.com/cutter/cut2.html
   
   
5. Scroll down until you see the box directing you to "Paste the DNA sequence into the box below." Place the cursor in the box. Paste the sequence into the box. Enter a title, if you wish.
   
   
6. Scroll down and click on the analyze sequence button.
   
   *Print the restriction map only (first 4 pages), and analyze.*
   
   (Don’t print the Table by Enzyme Name.)
   
   
7. Choose 1 restriction enzyme which cuts the target, is commercially available, and will generate visible products which can be detected using electrophoresis. (This will verify the product is the target from vanA gene.)
   
   
8. Use the RBASE database to retrieve information about your restriction enzyme.
   
   *Print the information for your chosen enzyme.*
   
   URL: http://www.neb.com/rebase/rebase.html
   
   Submit a report outlining the PCR procedure. It should include components of the master mix, method of product detection and verification using a restriction enzyme. Specific information about your chosen enzyme should be supplied. This should include organism source, recognition sequence (and cut) and the company where it can be purchased.

Top Objectives Overview Materials Procedures Extra Sites Questions Activities Evaluation

1. Master mix should include TAQ polymerase, dNTP (ATCG), template DNA, and 2 primers (left and right).
   
   
2. Detect product size of 159 bp using electrophoresis.
   
   
3. Chosen enzyme should cut in target area leaving 2 products large enough to visualize on agarose gel after electrophoresis.
   
   
4. Information concerning source organism, recognition sequence, and site of action; and commercial supplier, for chosen enzyme, is described.

Karen M. Kiser MA MT(ASCP), PBT
Emeritus Professor
Clinical Laboratory Technology/Phlebotomy
St. Louis, MO USA

Updated January 1, 2008

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