Principles of Genetic Engineering

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In Principle:
Genetic Engineering involves the laboratory manipulation of DNA
What does a particular region of DNA do?
    Isolation & manipulation of "gene of interest"

Restriction enzymes                                 [Nobel Prize 1978]
in vivo cloning"                                      [Nobel Prize 1980]
DNA sequencing                                       [Nobel Prize 1980]
Polymerase Chain Reaction (PCR)          [Nobel Prize 1993] 
CRISPR-Cas9                                             [Nobel Prize 2020]
Bioinformatics

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Sources of DNA
fresh viral, prokaryotic, or eukaryotic (plant & animal, etc.)
DNA separated from other molecules by selective binding
               (CSHL animation of DNA extraction)

Ancient DNA (aDNA)
museum specimens: 10s ~ 100s years
fossils: 1,000s ~ 1,000,000s years
Ex.: insects in amber
Ex.: ancient humans: Neanderthals, Denisovans, & relatives

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

Environmental DNA (eDNA)
                Ex.: What taxa (prokaryotic phyla or eukaryotic species) present?
                        Marine, freshwater, terrestrial monitoring

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In vivo Molecular Cloning of DNA

Type-II restriction endonucleases
     cut DNA only at specific restriction (recognition) sites  (list)
DNA palindrome -
             "Able was I ere I saw Elba"
             "Madam, I'm Adam"
             "A man, a plan, a canal: Panama!"
             "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."
             "Saippuakivikauppias" - 15-letter 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", "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 (unusual) Xgal sugar  blue product
lacZ gene includes polylinker
                    multiple, unique restriction sites

Recombinant DNA molecules formed when
linearization of vector DNA by digestion with endonuclease
ligation of "sticky ends" between source DNA & vector DNA
               re-combines DNA from two different sources

In vivo cloning in E. coli
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
Bacterium acquires genes from plasmid, including "Gene of Interest"

Colony Selection Scheme: finding 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 disrupts lacZ successful insertion & recombination !

Bulk bacterial culture of recombinant (white) colonies
           Purify cloned plasmid DNA, cut cloned gene out with endonucleases:
Gene of Interest ready for Analysis

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Polymerase Chain Reaction
        In vitro DNA "cloning": "DNA xeroxing" w/ four components & gadget

DNA template
            anything with DNA in it
       oligonucleotide primers ("oligos")
short (20 ~ 30 base) ssDNA complementary to gene of interest
                    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 ~ 80^oC, withstands exposure to 95^oC
dNTPs: four building-blocks for DNA
       Thermal cycler: computer-controlled heating & cooling block
                                   temperature change > 1^oC / 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]
            Thermal cycler automates process
replicates specific gene segment only
sufficient [DNA] for direct analysis

       Variations: quantitative PCR (qPCR)
            cf. qualitative (preparative) PCR produces DNA in bulk
quantitative (analytical) PCR rxns monitored in "real time"

Laboratory exercise in PCR primer design

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Analysis of cloned and (or) PCR-amplified DNA

Restriction mapping
        Determining the order of & distances among restriction sites in DNA fragment:
                provides an "outline" of the DNA sequence ["Mitochondrial Eve"]
                (much) finer scale than three-point test cross chromosome 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

Laboratory exercise in Restriction mapping

    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
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 single reaction
scanning laser activates & fluorometer "see" fluorescence colours (A C G T) [HOMEWORK]
                 computer "calls sequence" as chromatogram
                        (animation of automated DNA sequencing)

Next Generation sequencing uses massively-parallel, high-throughput methods
                NextGen sequencers use capillary separation, other methods

Laboratory exercise with DNA Sequences

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

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DNA animations on this page are available from Cold Spring Harbor Laboratory
     Click here to go to the download site

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All text material ©2025 by Steven M. Carr

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