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Lab Practical DNA Fingerprinting notes kcfac kilgore cc tx us gel


Lab Practical DNA Fingerprinting notes kcfac kilgore cc tx us gel

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									                      Biology 1408 Laboratory
               Inquiry–Based DNA Technology Laboratory
                      DNA Fingerprinting Notes

I. Learning Outcomes
       A. Student Should Be able to:
              --identify virus and viral infection
              --Identify hemorrhagic fever
              --Identify agarose gel
              --Identify electrophoresis
              --Identify restriction digest analysis
              --Identify DNA fingerprinting analysis

       B. The student must demonstrate successful completion of the laboratory
          exercise: Read Student Worksheet
             --cast agarose gel (20 pts)
             --load gel (20 points)
             --electrophoresis (20 pts)
       C. The student must submit results and conclusion
             --Describe the results: Compare the fingerprints of the
               Pennsylvania, Alabama, and Missouri virus strains. (20 pts)

               --What are your conclusions about the virus infecting the 3 Missouri
                 patients (20 pts)

II. What is the nature of viruses and viral infection?
      --viruses were first discovered by microbiologists seeking the cause of a plant
               --a nonliving, complex molecule
               --infectious particles, consist a protein coat ,containing nucleic acid of
                 RNA or DNA
               --protein coat on is called a capsid, encloses the genome
                       --some viruses also have an outer envelope

       --Viral Infection
       --viruses require a host cell to reproduce
       --inserts viral genome into that of host, transforming the normal cell into a
                  cancer cell
               --virus may remain lysogenic (dormant) or lytic, death of host cell

       --Virus and cancer
       --some groups can cause cancer: Retroviruses, papovavirus,
         adenovirus, ands herpesvirus

       --Emerging viruses
       --are not new but evolving, expanding into new species territories
       --sudden appearance in the late 1970s.
       --1976, Ebola in Ziare (central Africa)
               --virus caused hemorrhagic feve
               --often fatal, fever, vomiting, bleeding, collapse of circulatory
                 System. 4 decades this virus went unnamed.
       --1993, dozens died from hantavirus infection, southwestern U.S.
       --1995, Ebola (acute hemorrahagic fever)
               --central Africa, Zaire
       --1999, encephalitis, the Nipah virus in Malaysia, killed 105 people,
                destroyed the pig industry
       --2003, SARS (severe acute respiratory syndrome
               --pneumonia-like symptoms
               --natural host are a weasel-like mammal, Civets in China
               --other wild animals
               --meat is eaten as a delicacy

       --AIDS (acquired immune deficiency syndrome)
               --caused by the retrovirus, HIV
               --natural hosts are birds, migratory
               --new strains emerge each year
               --millions affected
               --possibly deaths

III. Three factors contribute to viral emergence
        --mutation of an existing viral strain
        --mutation of a strain which can cross between host species
        --exposure of an unknown viral agent to a larger area

IV. Basic health measures to prevent viral outbreaks
      --water purification
      --mosquito control
      --secure sewer systems

V. The use of biotechnlogy in the separation of macromolecules for identification
      A. gel electrophoresis is used
              --to separate molecules on basis of size and charge (+/-)
              --in forensic laboratories to help in solving crimes
              --to identify contagious and possibly deadly agents

VI. How gel electrophoresis works
      A. charged macromolecules migrate through a gel when placed in an electrical
             --smaller molecules migrate faster than larger ones

VII. What is the nature of the gels used in electrophoresis
      A. the gels contain the complex carbohydrate, agarose
               --extracted from seaweeds (red and brown algae)
               --a linear polymer
      B. the powered agarose is mixed in a buffer containing water, melted by heating
           To boil until clear (10-20 minutes).
               --allow to cool to 65oC before use
      C. the gel forms a matrix (contains small pores acts like a sieve).

IX. Restriction enzymes
      A. produced by bacteria
               --cut DNA molecules into smaller fragments
               --each enzyme recognize a specific restriction site
                           --most restriction sites are composed of 5-6 base pairs
      B. the restriction Eco RI was isolated from E.coli
               --recognition sequence                 :     ...………GAATTC………..

X. DNA Fingerprinting
     A. DNA molecules from all living organisms and viruses contain restrictions
     B. Restriction analysis can be used to identify a specific organism.

       C. Restriction analysis of specific sequences of DNA from different organisms
             --different size restriction fragments

       D. Small DNA fragments and viral genomes can be cut with
          restriction enzymes
               --electrophoresis produce a recognizable pattern
               --the patterns produced can be used to identify differences
                 in DNA sequences of organisms


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