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    [Nobel Prize 1980]
             analysis of the cloned gene
                   gel electrophoresis
                   DNA sequencing
                   mRNA expression
       Restriction enzymes                              [Nobel Prize 1978]
       DNA sequencing                                    [Nobel Prize 1980]
       Polymerase Chain Reaction (PCR)       [Nobel Prize 1993] 

Sources of DNA
       fresh viral, prokaryotic, or eukaryotic (plant & animal, etc.) material
             DNA separated from other molecules by selective binding to beads
               (CSHL animation of DNA extraction)
       Ancient DNA
                museum specimens: 10s ~ 100s years
                fossils: 1,000s ~ 1,000,000s years
                  Ex.: insects in amber
                  Ex.: ancient humans: Neanderthals & relatives

       Forensic DNA
            Forensics: data used as evidence
                Ex.: blood / semen stains at crime scenes
                Ex.: What species do these fillets come from?

In vivo Molecular Cloning of DNA

Type-II restriction endonucleases
     cut DNA only at specific restriction sites  (list)
      DNA palindrome -
             "Able was I ere I saw Elba"
             "Madam, I'm Adam"
             "Straw? No! Too stupid a fad, I put soot on warts."
             "A man, a plan, a canal: Panama!"
             ''Doc, note I dissent. A fast never prevents a fatness. I diet on cod."
             "Saippuakivikauppias" - Finnish word for a soap seller

      restriction site reads the same in 5'3' direction on both strands

    Overhanging TTAA-5' are "sticky ends"

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:
           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
                    multiple, unique restriction sites       
 
    Recombinant DNA molecules are formed when
           linearization of vector DNA by digestion with endonuclease
           ligation of "sticky ends" between source DNA & vector DNA
               combines DNA from two different sources  
           [online animation]

In vivo cloning in E. coli [IG1 02.18]
    E. coli K12 strain
          can't grow in presence of antibiotics (antibiotic-sensitive)
          can't metabolize X-galactose (Xgal) sugars
    Host transformation integrates plasmid DNA into bacterial chromosome
                 Bacteria acquires genetic traits of plasmid
  
    Colony Selection Scheme: 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
               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

Polymerase Chain Reaction
        In vitro DNA "cloning": [IG1 ResBrief 10.2, pp. 198-199]
         "DNA xeroxing": four components & a gadget

       DNA template
            anything with DNA in it
       oligonucleotide primers ("oligos")
            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
               denature / anneal / extend:  2  4  8  16  32  64 etc.:
                10 cycles = 210 = 103 copies, 20 cycles  106 copies, 30 cycles 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
 
Analysis of cloned and (or) amplified DNA

    Restriction mapping
        Determining the order of & distances among restriction sites in DNA fragment:
                provides an "outline" of the DNA sequence [IG1 21.22]
                (much) finer scale than three-point test cross map
       Gel electrophoresis separates DNA fragments by molecular weight
                          (animation of agarose gel electrophoresis)
           Fragment numbers & sizes compared in single & pairwise restriction digestions:
                  order & distances among sites determined as Restriction map
                          (CSHL animation)
           Restriction maps of overlapping fragments assembled as contig map


    DNA sequencing
          in vitro DNA replication copies one strand repeatedly
                                            (cf PCR: both strands replicated)
          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 [IG1 02.19]
             Sequencing gel shows series of partial DNA replications
                   Sequencing "ladder" autoradiogram read from bottom to top
                           (animation of dideoxy DNA sequencing)
  
       Automated DNA sequencers uses laser fluorometry
           ddNTPs attached to fluorescent dyes in a single reaction
                 scanning laser activates & fluorometer "see" fluorescence colours (A C G T) [HOMEWORK]
                 computer "calls sequence" as a chromatogram [IG1 02.21]
                        (animation of automated DNA sequencing)

        Next Generation sequencing uses massively-parallel, high-throughput methods [IG1 ResBrief 21.1, pp. 454-455]
                NextGen sequencers use capillary separation [IG1 02.20]


    Southern Blot analysis
        Useful when gene of interest rare: one locus / genome
        DNA transferred ("blotted") to filter paper
              Filter exposed to a DNA probe
                Probe: instrument or method that measures something (e.g., thermometer)
                            ssDNA complementary to gene region of interest
                             ~ same as primer / oligo in PCR experiment
                    Binds specifically to target DNA immobilized on filter
                    Radioactive label with  32P-dNTPs  exposes X-ray film
              Autoradiogram shows presence / absence & size of target DNA
                    RFLP differences

DNA animations on this page are available from Cold Spring Harbor Laboratory
     Click here to go to the download site
All text material ©2016 by Steven M. Carr