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 4 8
16
32 64 etc.:
10
cycles
=
103 copies, 20 cycles
106 copies,
30
cycles
(~2
hrs) 109
copies
[click
here
for
an 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
of agarose gel electrophoresis)
Fragment
sizes
in
single
&
pairwise
restriction
digestions compared:
order
& distances among sites determined
(Click here
for an 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
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 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 © 2024 by
Steven M. Carr