AP Biology (An Introduction)

							     Chapter 20


DNA Technology & Genomics
       Biotechnology Terms
 Biotechnology
       Process of manipulating organisms or their components to
        make useful products
       Genetic engineering + tissue/cell culturing technologies

 Genetic Engineering
       Manipulation of individual genes or entire genomes
       Insulin (insulin  E. coli bacteria OR yeast) & GMO
         (Genetically Modified Organism)

 Recombinant DNA
       Artificially created DNA
       Typically, DNA is integrated from another species

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Biotechnology Terms (Page 2)
 Gene Cloning
       Laboratory production of multiple copies of DNA segment
       Therapeutic cloning – embryonic stem cells
            Spinal cord injuries
       Reproductive (organismal) cloning – Dolly the sheep

 Restriction Enzymes
       Enzymes that cut DNA at specific locations
       Usually, derived from bacteria
       Cut sites of DNA = restriction fragments
       Sticky ends – restriction fragments usually have one end
         longer than the other


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            Quick Assignment

 Relate the 6 terms just discussed in a concept map.

 Be prepared to defend your arrangement




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               Cloning Process
 5 steps (first 2)
       1. Identify & isolate the gene of interest
           Involves finding a cloning vector – plasmid or organism used
             to carry the DNA sequence to be cloned


       2. Cut gene of interest from original site & open up vector’s
          DNA using a ________ ________
           This ensures matching sticky ends on gene of interest &
             vector DNA




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     Cloning Process (Page 2)
 5 steps (3-4)
      3. Combine the 2 DNA pieces (into a recombinant plasmid?)
          Recombinant plasmid – plasmid + DNA fragments
          Sealed together using DNA Ligase
          Remember: we used ________ ________ to cut gene of
            interest from original site & cut vector’s DNA
             This ensures matching sticky ends on gene of interest &
                vector DNA


      4. Transfer the vector (recombinant plasmid) into a host cell
          Usually involves bacterial transformation



                                                                        Slide 8 of 25
 Bacteria & Genetic Recombination
 Conjugation
    Bacterial Sex
    Genetic material is exchanged
     by direct contact



 Transduction
    Phage transfer of DNA
    Involves a phage vector
        Phage moves the DNA from
          bacterium to other bacterium


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Bacteria & Genetic Recombination

 Transformation
    Uptake of exogenous
     DNA
    Griffith’s experiment -
     pathogenic DNA was
     transferred to benign
     bacteria
    Most common method
     for genetic engineering




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                              Step 5
 Select for transformed cells
   Link the gene of interest with
      a reporter gene
   Such as pBLU or pGLO
   pBLU = Blue coloration
   pGLO = fluorescent green
      under UV light

    In Lab 6, we will insert the
       coloration gene and an
       ampicillin resistance gene to
       select for transformed cells

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                At this point…

 You know which cells have the gene of interest

 You can identify the cells that have the gene of interest

 Now what?

 You need to extract the gene of interest

 How would you do that?




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     Nucleic Acid Hybridization

 Detects the gene of interest

 Uses a short, single stranded DNA or RNA called a
   nucleic acid probe

 The nucleic acid probe is complementary to a known
   sequence in the gene of interest

 Usually attach a radioactive isotope or fluorescent tag
   protein so that it is detectable



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            Genomic Libraries

 Nucleic Acid Hybridization repeated many times
   produces a genomic library
    Thousands of recombinant clones
    Each has a piece of the original genome being studied




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                cDNA Library

 cDNA = complementary DNA

 mRNA is extracted from cells

 Use what enzyme to make DNA from this mRNA?

 Then make another strand of DNA using what enzyme?

 cDNA library is only a portion of the genome
    Portion that codes for mRNA
    Exons? Introns? tRNA? rRNA?



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               Microarray Assay

 Genome-wide study of gene expression

 Different genes are in each well

 Identifies gene interactions + provides clues to gene
   functions

 Take samples throughout development + assay to
   determine which genes are expressed and at what
   stages
    Detect patterns of expression throughout development
    Detect likely response to a pathogenic agent

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PCR
 Polymerase Chain Reaction
 Thermal cycling
 Amplification of DNA


3 Steps
 Denaturation (Heating)

 Annealing (Cooling)
   Primer formation

 Extension
     DNA polymerase adds
    nucleotides at 3’ end




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Gel Electrophoresis
                       DNA is negatively charged so it
                      moves AWAY from the (-) cathode
                      toward the (+) anode




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            Southern Blotting

 Used to detect specific DNA sequences

 Useful for comparing samples

 Combines gel electrophoresis + nucleic acid
   hybridization




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   DNA Technology affects us…

 Disease Diagnosis
    PCR used to detect traces of viral DNA or RNA in sample
    RFLP (Restriction Fragment Length Polymorphisms)
        Different alleles have different RFLPs

 Gene Therapy – alter afflicted genes

 Pharmaceutical Production – Insulin production

 Forensic Application – DNA fingerprints



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                        Page 2
 Environmental cleanup
    Genetically engineered microbes
    Detoxification of specific wastes

 Agricultural applications
    Insert pest-resistant or drought-resistant genes
    GMO (Genetically Modified Organisms)
        You eat GMO corn, soybeans, canola and cottonseed oil
        Probably at least weekly
        46% of GMOs are grown in US
        Europe had 12 year moratorium on growing GE foods


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