Principles of Genetic Engineering & Biotechnology
In Principle:
    Genetic Engineering involves the laboratory manipulation of DNA
          What does a particular region of DNA do?
     This may involve isolation & manipulation 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
       Restriction enzymes                                  [Nobel Prize 1978]
       DNA sequencing                                         [Nobel Prize 1980]
       Polymerase Chain Reaction (PCR)        [Nobel Prize 1993] 

For more details, see
        Bio4241 - Advanced Genetics (Dr Carr)
        Bio3950 - Fundamentals of Genetic Biotechnology (Dr Marshall)
Sources of DNA
       fresh viral, prokaryotic, or eukaryotic (plant & animal, etc.) material
             separation of DNA from protein & lipid with polar / non-polar solvents
             "kit" methods rely on selective binding of DNA

        Ancient DNA
                museum specimens: 10s ~ 100s years
                fossils: 1,000s ~ 1,000,000s years
                  Ex.: Magnolia fossil leaves at 18 MYBP
                  Ex.: insects in amber
                  Ex.: ancient humans: Neanderthals [iGen3, p. 236] and Oetzi the Iceman

       Forensic DNA of unknown or questioned origin
            Forensics: data used as evidence
                Ex.: blood / semen stains at crime scenes
                Ex.: What species do these fillets come from?
                Ex.: What is the "Sea Monster"?
 
       DNA Libraries
             Shotgun libraries by cloning (see below)
             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 [iGen3 8.15]

In vivo Molecular Cloning

Type-II restriction endonucleases
     cut DNA only at specific restriction sites  (list) [Igen3 Table 8.1]
      DNA palindrome -
             "Able was I ere I saw Elba"
             "Madam, I'm Adam"
             "Straw? No! Too stupid a fad, I put soot on warts."
             ''Doc, note I dissent. A fast never prevents a fatness. I diet on cod."

      restriction sites read the same in 5'3' direction on both strands [Igen3 8.1,2]

    Overhanging TTAA-5' "sticky ends" can recombine [Igen3 8.3]

Vector insertion
    Vector - a means of moving DNA from one place to another
       Plasmids - a circular, extrachromosomal DNA [Igen3 8.4]
                           "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 resistant (e.g., tetracycline or ampicillin)
                lacZ gene produces beta-galactosidase,
                    metabolizes Xgal sugar  "blue" product
                    lacZ gene includes polylinker       
 
    Recombinant DNA molecules are formed when
           Circular DNA is linearized by digestion with endonuclease
           ligation of "sticky ends" occurs between source DNA & vector DNA [iGen3 8.5]
               combines genes from two different organisms   [online animation]

Cloning in E. coli
    E. coli K12 strain - The prokaryotic wimp
          can't grow in presence of antibiotics (antibiotic-sensitive)
          can't metabolize X-galactose (Xgal) sugars
    Host transformation introduces plasmid into bacterial host
          Bacterial chromosome integrates plasmid DNA:
                 Bacteria acquires genetic traits of plasmid
  
    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 metabolize Xgal (Selection scheme)
               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
           Purify cloned plasmid DNA, cut cloned gene out with endonucleases:
           Gene is ready for Analysis

      SUMMARY OF pUC CLONING

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

Polymerase Chain Reaction
        In vitro DNA "cloning":
        "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 exposure to 95oC
       dNTPs: four building-blocks for DNA
       Thermal cycler: computer-controlled heating & cooling block
                                      temperature change > 1oC / sec

      PCR doubles gene copy number each cycle [iGen3 9.3]
          denature / anneal / extend:  2  4  8  16  32  64 etc.:
                10 cycles = 210 = 103 copies, 20 cycles  106 copies,
                    30 cycles (~2 hrs)  109 copies
                        [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 DNA

    Restriction mapping
        Determining the order of & distances among restriction sites in DNA fragment:
                this provides an "outline" of the DNA sequence
       Gel electrophoresis separates DNA fragments by molecular weight [iGen3 8.8]
           DNA is visible under Ultraviolet light with fluorescent dye
                          (animation of agarose gel electrophoresis)
           Fragment sizes in single & pairwise restriction digestions compared:
                  order & distances among sites determined
                          (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 [iGen3 10.8]
              Filter is exposed to a DNA probe
                Probe: instrument or method that measures something: e.g., thermometer
                             ~ same as single primer in PCR experiment
                    Binds specifically to target DNA immobilized on filter
                    Radioactively labeled with  32P-dNTPs  exposes X-ray film
              Autoradiogram shows presence / absence & size of target DNA
                    RFLP differences
                            (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) [iGen3 8.10]
                    stop strand growth during replication
                    four separate reactions terminate at ddA, ddC, ddG, or ddT [iGen3 8.11a,b]
               Sequencing gel shows series of partial DNA replications
                   Sequencing "ladder" autoradiogram is read from bottom to top
                           (animation of dideoxy 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 "calls sequence" as a chromatogram [iGen3 8.11c]
                Terra Nova Genomics Inc does this commercially
                        (animation of automated DNA sequencing)

        NextGen sequencing methods: massively-parallel Pyrosequencing, iGen3 8.12]

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

Homework Problems: MGA2 Chapter 8, pp. 259-260: ## 2, 3, 5, 6, 7, 13
All text material © 2011 by Steven M. Carr