Download Document

Biotechnology
What does it mean?
Tools and Technologies
Selected Applications
Biotechnology 1: any method based on knowledge of
biological processes that achieves a practical purpose for
human civilization; 2: applied biology.
Biotechnology Past and Present:
• Early Civilizations:
– Domesticated plants and animals
– Preservation of food
• beer, mead, wine
• fermented vegetables
• cheese, yogurt
– Bread!
•
Emergence of Modern Biotechnology:
– Mendelian genetics and hybrid organisms (e.g., crop plant advances)
– Microbial fermentation; acetone and glycerol for WWI ammunitions.
– Fleming’s discovery of antibiotics saved lives in WWII.
– Watson and Crick described DNA (won Noble prize)
•
Modern Genetic Engineering: (ability to create recombinant DNA in lab)
– produce medications (hormones; antibiotics)
– make disease / drought resistant plants
– identify genetic material to help solve crimes (forensics)
– clean up of toxic spills (Exxon Valdez)
– whole-organism cloning of livestock species
Scope of Modern Biotechnology
(Genetic Engineering)
Be able to recognize and group the examples we discuss under each of these
six categories; i.e. match discussed examples with categories.
Gene Cloning Overview:
How is DNA cut
at known sites?
Artificial transformation is
used to get plasmids back
into a host bacterium.
How will we select for only
transformed bacteria with
recombinant plasmids?
How do we know
which is to be cloned?
1) Clone all genome
fragments and find
what you want from
this library afterwards.
2) Find the desired
DNA fragment before
cloning.
How is DNA cut at known sites?
Restriction endonucleases are enzymes bacteria make to cut foreign DNA (like that
from an infecting virus). Each species of bacteria has a “restriction enzyme” that cuts
DNA at a unique “palondromic” sequence of 4 to 8 base pairs, called recognition sites.
Cutting of each strand is often unevenly, which create overhanging ends of single
strand DNA, called “sticky ends”
When cloning genes, we cut
the DNA of the foreign
donor and that of the
plasmid vector using the
same restriction enzyme.
This creates the same
sticky ends on both vector
and fragment DNA.
A ligation reaction refers to
the mixing, annealing of
sticky ends and ligase
reaction to form
recombinant plasmids.
In ligation reactions, not all
plasmid DNA will have
foreign DNA fragments
inserted. Nor will all
bacteria receive a plasmid
after transformation.
Plasmids Designed for Cloning Genes:
• A plasmid vector should be designed to have genes for easily recognized
phenotypes that can be used to distinguish the three different bacterial populations
that result after artificially transforming a host bacterial culture.
• Non-transformants = host bacteria
that did not receive any plasmid.
• Non-recombinant transformants =
host bacteria with a normal plasmid
vector (no inserted foreign DNA).
• Recombinant Transformants =
host bacteria with recombinant
plansmid (has inserted foreign DNA).
• Antibiotics, like ampicillin, in the agar
media post-transformation will prevent
growth of non-transformants.
• Genes on plasmids that can
obviously change colony appearance
(e.g. lacZ) are good sites for inserting
foreign DNA into plasmid vector DNA.
“Cloning site”
Cut and
insert here
Blue-White Colony Selection:
3) Artificial transformation of
host bacterium (Amp sensitive).
1) Cut inside lacZ
with same restriction
enzyme used to cut
foreign donor DNA.
4) Plate to Amp+ media with the
β-galactosidase substrate analog.
Non-recombinants have functional
enzyme; their colonies turn blue.
Recombinant colonies are white.
2) Ligation reaction.
How to clone the gene you want?
Create a “Library”:
•Clone enough fragments
of foreign donor DNA to
represent the entire
genome of the organism of
interest.
• Each clone will represent
a portion of the genome.
• Libraries may use
plasmid vectors and host
bacteria, or they may use a
bacteriophage vector.
• The library can then be
screened for any gene of
interest, and used over and
over again.
Screening a Library by Colony Hybridization
Gene probes: Small DNA sequences
(oligonucleotide) complementary to only
the gene of interest that is “labeled” for
detection, using radioactivity in this case.
Agarose Gel Electrophoresis:
Separates DNA by size; many applications, including “fingerprinting” and screening
fragments of foreign donor DNA with a particular gene for cloning.
A fluorescent DNA
stain is used to see
bands under UV light.
Southern Blot
Can’t hybridize DNA probe
to sample DNA in a gel.
Water and some DNA is blotted (or wicked)
out of the gel; DNA gets trapped and binds
to nitrocellulose paper. Now we can “probe”.
The only thing
you can see!
DNA fragment detected by probe can be
cut from gel and then cloned.
Polymerase Chain Reaction (PCR):
Specific target sequences of DNA (e.g., gene for cloning) found in very low levels
in a sample can be amplified to billions of copies to use in further manipulation
(e.g., gene cloning, DNA fingerprinting, genetic screening).
•
Repeated cycles of replication for a
specific DNA sequence will
exponentially increase copies on only
that DNA sequence. N=2n
•
Each cycle has 3 major steps:
1) Denaturing DNA from helix to
single strands.
2) Annealing primers; one specific to
each end of the target DNA
sequence.
3) Extension of new DNA strand by
a heat tolerant DNA Polymerase
(from a thermophilic bacterium)
Recombinant Protein Products
Transgenic Plants