Chapter 20

Recombinant DNA
technology,
Genetic engineering
and
Biotechnology

A few definitions
Biotechnology
Manipulation of organisms to make useful products

Genetic engineering (one part of Biotechnology)

Manipulation of genes (mostly for practical purposes ie: to
make something we want)

Recombinant DNA
DNA from different sources are combined in vitro (in the
lab)

DNA cloning
Isolate a gene (of interest) and make multiple copies

Plasmids
Small, circular pieces of bacterial DNA used in
biotechnology to move genes between organisms

DNA cloning
Plasmid from a
bacterium
+
Gene of interest
=
recombinant DNA
plasmid

Put into and

grow bacteria
to produce
LOTS of the gene
product
(protein)

Cloning a eukaryotic gene into a

bacterial plasmid (cloning vector)
Cut DNA (plamid and DNA of interest) with restriction
enzymes
Mix
cut
together
cut

Ligase seals the strands together

Plasmid with an insert is a recombinant plasmid

DNA cloning
The big (and simple) picture

How restriction
enzymes work

Put the plasmid into bacteria

Electroporation - give a jolt of electricity and
bacteria will take up DNA
Ampicillin resistance gene on the plasmid
allows you to select only the bacteria that
successfully picked up the plasmid
Bacteria containing plasmid are AmpR
Bacteria without plasmid are AmpS

Grow the bacteria so they will express

the gene of interest
(make the protein you want)
More complicated than it sounds!

DNA cloning
Plasmid from a
bacterium
+
Gene of interest
=
recombinant DNA
plasmid

Put into and

grow bacteria
to produce
LOTS of the gene
product
(protein)

How can we obtain the

gene of interest for
cloning?
1. Genomic DNA
cut
cut

2. PCR

PCR

PCR

Cycle 20
yields 1
million
molecules
!

DNA fragments made by PCR can

be cut with restriction enzymes
for use in DNA cloning

DNA cloning allows us to introduce

new genes into cells
How can we study the function and
expression of genes within cells?

Studying gene expression

Nucleic acid hybridization
Uses labeled Nucleic acid probes
ie: If part of the desired gene had the
following sequence:

Then the complimentary labeled probe would

look like this

Studying gene expression by

in situ nucleic acid
hybridization

We can use
multiple labeled
probes
simultaneously:

Only cells that

express the
wingless (wg)
gene will bind the
yellow probe
Only cells that
express the
engrailed (en)
gene will bind the
blue probe

Biotechnology has many

potential applications
Production of
protein
products
HGH
Human insulin
Vaccines

Stem cells
Gene therapy
Agricultural
applications