Clinical Laboratory Technology
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.
TITLE OF LESSON: Tools of DNA Analysis
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.
- Describe the tools used to analyze DNA
- Design a PCR procedure which detects and verifies the presence of a specific gene.
- Relate information about source organism, action, and supplier for restriction
- Computer with Internet access
- Netscape Browser with Shockwave plug-in
To learn more about monoclonal antibody technology, go the following sites.
- Read about the Language of Recombinant DNA.
- View an overview of DNA manipulation.
- Learn more about restriction enzymes by reading the article and viewing images at
- The Polymerase Chain Reaction is described at these sites.
Access Excellence: Polymerase Chain Reaction-Xeroxing DNA
Access Excellence: Polymerase Chain Reaction
- Learn more about recombination at these sites:
- Read the section on Sequence Technologies in the Primer on Molecular Genetics.
- See animations of PCR, Southern Blots, and Cycle Sequencing at the Biology
- Identify an unknown bacterium from the base-pair sequence using ribosomal DNA, at the Virtual Bacterial ID Lab.
To learn more about the practical applications of biotechnology, visit the following site.
To learn more about genome projects, visit the following site.
Each student will design a PCR procedure to detect an antibiotic resistance gene.
- Once a target sequence is amplified, how will you know whether itís present?
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.
A report outlining PCR procedure should meet the following criteria:
- Use the European Bioinformatics Institute site to search and retrieve the nucleic acid sequence for the Enterococcus
- 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
- View and copy the DNA sequence.
- Open a second browser and go to the Primer 3 site to choose primers.
Place the cursor in the box. Paste the sequence into the box and click "pick primers."
*Print out the results.*
- To generate a restriction map of the target area, go to the following address:
- 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.
- 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.)
- 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.)
- Use the RBASE database to retrieve information about your restriction enzyme.
*Print the information for your chosen enzyme.*
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.
© Karen M. Kiser 1998
- Master mix should include TAQ polymerase, dNTP (ATCG), template DNA, and 2
primers (left and right).
- Detect product size of 159 bp using electrophoresis.
- Chosen enzyme should cut in target area leaving 2 products large enough to visualize
on agarose gel after electrophoresis.
- 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
Clinical Laboratory Technology/Phlebotomy
St. Louis, MO USA
Updated January 1, 2008
Comments or suggestions?