Principles of
Genetic Engineering
In Principle:
Genetic Engineering involves the laboratory
manipulation of DNA
What does a particular
region of DNA
do?
Isolation & manipulation of "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 & 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 species (prokaryotic or eukaryotic)
present?
Marine, freshwater, terrestrial
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", 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
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
Bacteria acquires genetic traits of plasmid
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 is ready for Analysis
Polymerase
Chain Reaction
In vitro DNA "cloning": "DNA
xeroxing": four components & a 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 ~ 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
[
of PCR]
PCR process automated
replicates specific gene
segment only
sufficient [DNA] for direct analysis
Variations: quantitative
PCR (qPCR)
cf.
preparative PCR: individual rxns monitored in
"real time"
Laboratory exercise in PCR primer design
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 ["Mitochondrial Eve"]
(much) finer scale than three-point test cross map
Gel electrophoresis
separates DNA fragments by molecular weight
( 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
(
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
( of automated DNA sequencing)
Next
Generation sequencing uses massively-parallel,
high-throughput methods
NextGen sequencers use capillary separation
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
DNA animations on this page are available from Cold
Spring Harbor Laboratory
Click here to go to the download
site