Biotechnology by l7lSz7Oc


  Chapter 14
              DNA Manipulation

•Cloning is the production of identical copies of DNA through
some asexual means.
    •An underground stem or root sends up new shoots that
    are clones of the parent plant.
    •Members of a bacterial colony on a petri dish are clones
    because they all came from division of the same cell.
    •Human identical twins are clones; the original single
    embryo separate to become two individuals.
              DNA Manipulation
•Gene cloning is production of many identical copies of the
same gene.
   •If the inserted gene is replicated and expressed, we can
   recover the cloned gene or protein product.
   •Cloned genes have many research purposes:
   determining the base sequence between normal and
   mutated genes, altering the phenotype, obtaining the
   protein coded by a specific gene, etc.
   •Humans can be treated with gene therapy: alteration of
   the phenotype in a beneficial way
                    DNA Manipulation
• Recombinant DNA (rDNA) contains DNA from two or more different
    – Requires:
        • A vector
             – introduces rDNA into host cell
             – Plasmids (small accessory rings of DNA from bacteria) are common vectors
             – Phage vectors (bacterial viruses) can also be used
        • Two enzymes to introduce foreign DNA into vector DNA
             – A restriction enzyme - cleaves DNA
                  – Bacterial enzyme that stops viral reproduction by cleaving viral DNA
                  – Act as molecular scisssors (cut plasmids and foreign human DNA)
                  – Produce short single stranded “sticky ends” where insertions of foreign
                     DNA can be made
             – A DNA ligase enzyme - seals DNA into an opening created by the restriction

Cloning a Human

        Restriction enzyme EcoRI
    –Bacterial enzyme that stops viral reproduction by
    cleaving viral DNA
    –Act as molecular scisssors (cut plasmids and
    foreign human DNA)
    –Produce short single stranded “sticky ends”
    where insertions of foreign DNA can be made

          Genetic Engineering

• Has generated excitement and controversy

• Transgenic organisms have had a foreign gene
  inserted into their genetic make-up

       Agricultural Applications
Ti (tumor-inducing) plasmid is the most used
  vector for plant genetic engineering
  -Obtained from Agrobacterium tumefaciens,
  which normally infects broadleaf plants
  -However, bacterium does not infect corn, rice
  and wheat

Agricultural Applications

        Agricultural Applications
Herbicide resistance
 -Broadleaf plants have
 been engineered to be
 resistant to the herbicide

  -This allows for no-till

       Agricultural Applications
Pest resistance
  -Insecticidal proteins have been transferred into
  crop plants to make them pest-resistant
      -Bt toxin from Bacillus thuringiensis

Golden rice
 -Rice that has been genetically modified to
 produce b-carotene (provitamin A)
     -Converted in the body to vitamin A
       Agricultural Applications
Adoption of genetically modified (GM) crops has
  been resisted in some areas because of
  questions about:

  -Crop safety for human consumption
  -Movement of genes into wild relatives
  -Loss of biodiversity

       Agricultural Applications
  -Transgenic plants are used to produce
      -Human serum albumin
      -Recombinant subunit vaccines
        -Against Norwalk and rabies viruses
      -Recombinant monoclonal antibodies
        -Against tooth decay-causing bacteria
     Transgenic Mammals

         Medical Applications

• The insertion of genetic material into
  human cells for the treatment of a

Recombinant DNA Vaccines?

   Strategy for a subunit vaccine for herpes simplex
                       Gene Therapy

Treatment of SCID (severe combined immunodeficiency). SCID affects the
maturation of immune cells that develop in bone marrow. SCID sufferers lack the
enzyme ADA (adenosine deaminase).
                    DNA Analysis
Polymerase chain reaction (PCR)
  -Developed by Kary Mullis (1983) (got Noblel)
  -Allows the amplification of a small DNA fragment using
  primers (supplied nucleotides) that flank the region
  -Allows thousands of copies to be made of small samples
  of DNA
  -Each PCR cycle involves three steps:
   1. Denaturation (high temperature)
   2. Annealing of primers (low temperature)
   3. DNA synthesis (intermediate temperature)
        -Taq polymerase (DNA polymerase)

               DNA Analysis
Polymerase chain reaction (PCR)
  -Has revolutionized science and medicine
  because it allows the investigation of minute
  samples of DNA
     -Detection of genetic defects in embryos
     -Analysis of mitochondrial DNA from early
     human species and other animals

             Applications of PCR:
Analyzing DNA Segments via Gel Electrophoresis
• DNA can be subjected to DNA fingerprinting
     – Treat DNA segment with restriction enzymes
        • A unique collection of different fragments is produced
        • Gel electrophoresis separates the fragments according
          to their charge/size
        • Produces distinctive banding pattern
• Usually used to measure number of repeats of
  short sequences
• Used in paternity suits, rape cases, corpse ID,

            Gel Electrophoresis
• A technique used to separate DNA fragments by
• The gel (agarose or polyacrylamide) is subjected
  to an electrical field
• The DNA, which is negatively-charged, migrates
  towards the positive pole
  – The larger the DNA fragment, the slower it will
    move through the gel matrix
• DNA is visualized using fluorescent dyes
            DNA Fingerprinting & Paternity

     Male 1 is the father. His DNA fingerprint is like that of the child except
     for the portion supplied by the mother.

            DNA Analysis

Two of the DNA profiles used to convict Tommie Lee
Andrews for rape in 1987 .

           Human Genome Project
• Genome - All the genetic information of an
  individual (or species)
• Goals of Human Genome Project (completed
     – Determine the base pair sequence in all human DNA
        • 13 year project
        • Working draft
     – Construct a map showing sequence of genes on
       specific chromosomes
• Other species in final stages

Partial map for the human X chromosome (pg 246)

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