Principles of Genetic Engineering & Biotechnology

In Principle:
    Genetic Engineering involves the laboratory manipulation of DNA (MGA2 8-2)
          What does a particular region of DNA do?

    This may involve isolation of a "gene of interest"
             in vivo or in vitro "cloning" of the gene
             analysis of the cloned gene
                   gel electrophoresis
                   nucleic acid "blotting"
                   DNA sequencing
                   mRNA expression

    Biotechnology: “The use of biological systems to create goods & services"
       Restriction enzymes                                  [Nobel Prize 1978]
       DNA sequencing                                         [Nobel Prize 1980]
       Polymerase Chain Reaction (PCR)        [Nobel Prize 1993] 

For more details, see
        Bio2250 - Principles of Genetics

    DNA sequencing

          in vitro DNA replication reaction copies one strand repeatedly
          Provides complete order of bases in a DNA fragment
       DNA primer is complementary to 3' end of gene of interest
                    primers are end-labelled with 32P or 35S
       dideoxynucleotide terminators (ddNTPs) (MGA2 8-23)
                    stop strand growth during replication
                    four separate reactions terminate at ddA, ddC, ddG, or ddT (MGA 8-22a)
               Sequencing gel shows series of partial DNA replications
                   Sequencing "ladder" is read from bottom to top in an autoradiogram
(MGA2 8-22b)
           [click here for an
animation of autoradiographic DNA sequencing]

       Automated DNA sequencer uses laser fluorometry
           ddNTPs are attached to fluorescent dyes
                scanning laser & fluorometer "see" fluorescence colours (A C G T)
                computer compares colours, "calls sequence" (MGA2 8-24)

                Alternative separation methods: Capillary electrophoresis

            [click here for an animation of automated DNA sequencing]
      Bio4241 - Sanger et al. 1977

Microarray DNA sequencing -  "DNA Chips"
    DNA Re-Sequencing Chips for iterative sequencing of populations [PPT presentation]
The ArkChip for multiplex species populations studies

All text material © 2010 by Steven M. Carr