Genetic Engineering

Recombinant DNA (rDNA) Technology

• rDNA technology
involves cloning DNA by
cutting & pasting DNA
from different sources
• Restriction enzymes &
DNA ligases are
important enzymes for
this process
• DNA ligases join together
adjacent DNA fragments
 Genetically Modified Organisms
(GMOs)
• GMOs are organisms that have had
genetic material removed and/or
inserted in order to change a
particular trait or traits of the
organism.
• The process is called gene splicing
or genetic engineering
• Organisms produced by
transplanting genetic materials
between different types of organisms
are called transgenic organisms.
Transgenic Organism Examples
• Genes from bacteria are spliced into
corn and cotton to make them less
susceptible to insect damage
• Human growth hormone implanted
into mice & other animals so that it
can be harvested
• ANDi (first transgenic monkey) is a
rhesus monkey carrying GFP protein,
showing foreign gene can be inserted
into primate chromosome
• May lead to primate models of
human diseases
Restriction enzymes
• Restriction enzymes are DNA-
cutting enzymes that are found in
bacteria
• They are also called
endonucleases (cut within DNA
sequences)
• Microbiologists from 1960s
discovered that some bacteria are
protected from destruction by
viruses because they cut viral DNA,
restricting viral replication
 Restriction enzymes Q & A

In 1970, Hamilton Smith isolated HindIII (1st restriction

Whichwell
enzyme was the first one
characterized andwell
usedunderstood?
for DNA cloning),
which comes from Haemophilus influenzae.

They are named based on genus & species of bacteria

How are they named?
it was isolated from. (EcoRI = Escherichia coli, RY13).

They cut DNA by cleaving phosphodiester bonds (in

How
sugar-phosphate do they
backbone) work?
that join adjacent
nucleotides
Specificity
• Restriction enzymes show specificity
for certain substrates (DNA in this case)
• They recognize, bind to, and cut DNA
at specific sites called restriction sites
(recognition site)
• Usually a 4-base pair or 6-base pair
cutter
• Restriction sites are palindromes
(reads same forward & backwards on
opposite strands)
 Restriction cuts
• Some cut DNA to create
fragments with overhanging
single-stranded ends (sticky
ends or cohesive ends),
while others create
fragments with non-
overhanging ends (blunt
ends)
• Enzymes that create sticky ends are favored for cloning
experiments since the DNA fragments can be easily joined
together
• DNA from any source can be digested (as long as it has the
specific restriction site)
GE Application
• In 1972, Paul Berg joined
DNA from E.coli and a primate
virus called SV40
• He cut both with EcoRI
(restriction enzyme)
• He then added fragments to
tube with DNA ligase
• This became 1st
recombinant DNA molecule
 Plasmids
• Plasmid DNA is circular
form of self-replicating DNA
that scientists can
manipulate to carry and
clone other pieces of DNA
• Found primarily in
bacteria
• Considered extrachromosomal DNA because they are
present in addition to chromosomes
• They are small (~1000 - 1400 base pairs) in size
Vectors
• Plasmids can be used as vectors
(pieces of DNA that can accept,
carry, and replicate other pieces of
DNA)
• 1st plasmid vector pSC101
(SC = Stanley Cohen, pictured left)
• Contained gene for tetracycline
(antibiotic) resistance and restriction
sites for several enzymes
• rDNA animation
Vectors
• Cohen & Boyer (pictured left)
awarded patents (1980) for pSC101
and gene splicing & cloning
technologies
• Major concern at the time was the
thought of recombinant bacteria
leaving the lab
• Boyer joined forces with Robert
Swanson (venture capitalist) to
create Genentech in an effort to
commercialize these technologies
 Vector Features

Modern plasmid DNA cloning

vectors usually consider 6
desirable features:
1. Size (must be small enough
to separate easily)
2. Origin of replication (ori) -
DNA sequence at which
replication is initiated
3. Multiple cloning site (MCS) - a stretch of DNA with recognition
sequences for common restriction enzymes (Engineered into
plasmid so that digestion does not result in loss of DNA fragment)
 Vector Features
4. Selectable marker genes - allow for selection and
identification of transformed bacteria
• Most common selectable markers
are antibiotic resistance.
• Lac z gene widely used (gene of
interest inserted within lac z gene)
• Plated on X-gal (substrate similar
to lactose but turns blue when
cleaved by ß-gal); so, recombinant
bacteria turn blue &
nonrecombinant are white
Selection
• Selection is a screening
process designed to facilitate the
identification of recombinant
bacteria while preventing growth
of nontransformed bacteria (or
those containing plasmid without
foreign DNA)
• Blue-white screening is
becoming more popular (uses ß-
galactosidase)
 Antibiotic selection
• Antibiotic selection uses a
plasmid vector with genes
encoding resistance to 2
different antibiotics, usually
ampicillin (ampR) and
tetracycline (tetR)

• Foreign DNA inserted into one of the 2 antibiotic

resistance genes (disrupts gene - preventing protein)
• Transformed cells are plated to an agar plate with no
antibiotic or plate with one (ampicillin)
 Replica plating
• Replica plating uses
sterile pads pressed against
colonies on plate (cells
adhere to make an exact
copy)
• Then pad is placed on 2nd
replica plate containing 2nd
antibiotic (tetracycline)
• Nontransformed bacteria cannot grow in presence of either
antibiotic without plasmid
• Compare plates since recombinant can’t grow on 2nd plate
Replica plating diagram
 Vector Features

5. RNA polymerase promoter

sequences - place where RNA
polymerase binds to begin
transcription
6. DNA sequencing primer
sequences - known sequence
that allows sequencing of
cloned DNA fragments that
have been inserted into the
plasmid
 Types of Vectors
One primary limitation of bacterial plasmids as vectors is
the size of DNA fragments (usually cannot exceed 6-
7kb: 6000-7000 base pairs).
• Bacteriophage vectors
• Expression vectors
• Bacterial artificial chromosomes (BACs)
• Yeast artificial chromosomes (YACs)
• Tumor-inducing (Ti) vectors
 Gene Transfer
• Cohen discovered that plasmid
DNA enters a bacterial cell
(transformation) treated with
calcium chloride, chilled on ice,
then briefly heated
• A more recent transformation
technique is electroporation (brief
pulse of high-voltage electricity to
create tiny holes in bacterial cell
wall allowing DNA to enter)
• Cells that have been treated for transformation (so they are
more receptive to take up DNA) are called competent cells
 Biolistics
• Sometimes, biolistics are used
in order to have foreign DNA enter
a cell
• DNA is blasted into the cell using
tiny bullets composed of tungsten
or gold particles with DNA
attached
• Done with a gene gun (aka
bioblaster)
• Can be used on bacteria, yeasts,
& mammalian cell lines
 National Institutes of Health
(NIH)
• Concerns arose
because of new
techniques
• In 1975, NIH formed
the Recombinant DNA
Advisory Committee
(RAC) to evaluate risks
and establish guidelines
for rDNA technology