Primer of Genetic Engineering & Biotechnology

In Principle:
    Genetic Engineering involves the laboratory manipulation of DNA
    This may involve isolation of a "gene of interest"
             in vivo replication or in vitro "cloning" of the gene
             analysis of the cloned gene
                   gel electrophoresis
                   nucleic acid transfers "Southern blotting"
                   DNA sequencing

     What does a particular region of DNA do?
             Reverse Genetics: DNA   RNA
  Protein phenotype
             Bioinformatic comparison with other DNA sequences


    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
        Bio4241 - Advanced Genetics
        Bio2250 - Principles of Genetics ]


Sources of DNA
       fresh viral, prokaryotic, or eukaryotic material
             separation of DNA from protein & lipid with polar / non-polar solvents
             "kit" methods: selective binding of DNA

       Ancient DNA
                museum specimens: 10s ~ 100s years
                fossils: 1000s ~ 1,000,000s years
                  Ex.: Magnolia at 18MYBP

                  Ex.: insects in amber

       Forensic DNA
of unknown or questioned origin
                blood / semen stains
                "The Case of the Falsified Fillets"
                "The Case of the Scurrilous Scallops"
                "How to Tell a Sea Monster"

       DNA Libraries

             Expression libraries
                 mRNA
from gene of interest  may be abundant  in a particular tissue
                     Ex.: Lysozyme in abomasum stomach of ruminants
                     Ex.: glue protein in salivary glands of larval Drosophila
                mRNA cDNA (complementary DNA) via reverse transcriptase
                        cDNA will have exons only


Molecular Cloning & Recombinant DNA

Type-II restriction endonucleases

     cut DNA only at specific restriction site  (expanded list)
      DNA palindrome -
             "Able was I ere I saw Elba"
             "Madam, I'm Adam"
      restriction sites read the same in 5'3' direction on both strands

    Overhanging TTAA-5' "sticky ends" can recombine

Vector insertion

    Vector - a means of moving DNA from one place to another
       Plasmids - a circular, extrachromosomal DNA,
                           "naked DNA", a "bacterial virus"
       pUC18 - an artificial plasmid with:
           polylinker containing multiple, unique restriction sites
           selectable markers that tell you when plasmid is present
               antibiotic resistance (e.g., tetracycline or ampicillin)
                lacZ gene produces beta-galactosidase,
                    metabolizes Xgal sugar  "blue" product
    Recombinant DNA molecules are formed when
         ligation of "sticky ends" occurs between source DNA & vector DNA
             combines genes from two different organisms   [online MGA2 animation]

Cloning in E. coli

    E. coli K12 strain - The prokaryotic wimp
          antibiotic-sensitive
          can't metabolize galactose (Xgal) sugars
    Host transformation introduces plasmid into bacterial host
    Colony Selection: finding the rare bacterium with recombinant DNA
          Only E. coli cells with resistant plasmids grow on antibiotic medium
            only plasmids with functional lacZ gene can grow on Xgal
               lacZ(+) => blue colonies
                   lacZ functional => polylinker intact => nothing inserted, no clone
               lacZ(-) => white colonies
                    polylinker disrupted => successful insertion & recombination!

    Bulk bacterial culture of recombinant white colonies
           Bacterial replication also replicates recombinant plasmid
           Purify cloned plasmid DNA,
                Gene is ready for Analysis

 
     SUMMARY OF pUC CLONING

     
The Cartoon Guide to Molecular Cloning  (Gonick & Wheelis 1991)


In vitro DNA "cloning": The Polymerase Chain Reaction
        "DNA xeroxing": four components & one gadget

       DNA template
            anything with DNA in it
       oligonucleotide primers
            short (20 ~ 30 base) ssDNA complementary to gene
                    some knowledge of gene is required :
                       "Universal primers" work across many species
       Taq DNA polymerase
                    heat-stable enzyme from hot-spring bacteria (Thermus aquaticus)
                    functional at 70 ~ 80oC, withstands repeated exposure to 95oC
       dNTPs: four building-blocks for DNA
       Thermal cycler: computer-controlled heating & cooling block

      PCR doubles gene copy number each cycle 
          denature / anneal / extend:  2  16  32  64 etc.:
                10 cycles = 103 copies, 20 cycles  106 copies,
                    30 cycles (~2 hrs)  109 copies
                [click here for an animation of PCR] [online MGA animation]
            PCR process is completely automated
                      replicates specific gene only
                      makes sufficient quantities of purified genes for direct analysis
                           [mtDNA gene in RFLP Lab amplified by PCR]
 


Analysis of cloned DNAs
    Is the one you wanted?
    What are its molecular characteristics?

    Restriction mapping
        Determining the order of restriction sites in a cloned fragment:
                this provides an "outline" of the DNA sequence
       Gel electrophoresis separates DNA fragments by molecular weight
           DNA is visible under Ultraviolet light with fluorescent dye
                  (Click here for an animation of agarose gel electrophoresis)
           Fragment sizes in single & pairwise restriction digestions compared:
               order & distances among sites determined 
                  (Click here for an animation of Restriction Mapping logic)
           Restriction maps of adjacent fragments assembled as contig map

    Southern Blot analysis

        Useful when gene of interest is rare: one locus / genome
        DNA is transferred ("blotted") to filter paper
              Filter is exposed to a DNA probe
              Probe - single-stranded DNA, base-complementary to gene of interest
                    Binds specifically to target DNA immobilized on filter
                    Radioactively labeled with  32P 35S-dNTPs  exposes X-ray film
              Autoradiogram shows presence / absence & size of cloned DNA
                    RFLP differences
                    [click here for an animation of southern blotting]
       DNA probe binds to RNA: "Northern Blot"
                 Is a particular gene (DNA) expressed as mRNA in a particular tissue?
            [Antibody probe binds to protein: "Western Blot"]

    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
            dideoxynucleotide terminators (ddNTPs)
                    stop strand growth during replication
                    four separate reactions terminate at ddA, ddC, ddG, or ddT
               Sequencing gel shows series of partial DNA replications
       Sequencing "ladder" is read from bottom to top

       Automated DNA sequencer uses laser fluorometry
           ddNTPs are attached to fluorescent dyes
                scanning laser & fluorometer "see" fluorescence colours (A C G T)
                computer assembles four colours, "calls sequence"
                "Helix & Primer" DNA Sequencing Lab does this at MUN
            [click here for an animation of automated DNA sequencing]


DNA animations on this page are available from Cold Spring Harbor Laboratory
     Files are in PC or Mac format & require the Shockwave viewer:
     Click here to go to the download site


All text material © 2010 by Steven M. Carr