St. Louis Community College

Clinical Laboratory Technology



INTERNET LESSON PLAN NUMBER 3

UNIT - Molecular Techniques

SCOPE OF UNIT:

Review nucleic acid structure and function. Relate the tools of DNA analysis to include the use of the Internet. Use molecular techniques and Internet databases to analyze genetic material.

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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:

The tools of DNA analysis include enzymes and fundamental procedures which allow amplification and detection of specific targets on the genome and the construction of genome libraries using recombination techniques. Much of the information we know about the genetic make-up of humans, various animals and microorganisms is available on the Internet in databases. With this knowledge, diagnostic procedures can be developed to identify specific genes and nucleic acid sequences of microorganisms. Therapy for genetic diseases will also be devised in the future.

MATERIALS:

PROCEDURES:

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  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/

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EXTRA SITES OF INTEREST

To learn more about monoclonal antibody technology, go the following sites.

To learn more about the practical applications of biotechnology, visit the following site.

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

To learn more about genome projects, visit the following site.

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

INTERACTION ITEMS:

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

CLASSROOM, LABORATORY, OR OTHER ACTIVITIES

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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.*

    (Donít 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.
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EVALUATION:

A report outlining PCR procedure should meet the following criteria:

  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 1998


St. Louis Community College at Forest Park
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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