Download Genetic Engineering (and other cool molecular biology techniques)

Genetic Engineering
(and other cool molecular biology techniques)
DNA Cloning
• DNA cloning permits production of multiple
copies of one particular gene
• This gene copy will be used to produce a usable
protein product (e.g. insulin) or for genetic
engineering (e.g. gene for pest control inserted
into plant)
For Fun:
• We want to clone a gene that produces a
protein that will help you focus while
studying.
1. We need the gene
• This gene is located on chromosome 16
2. We need a vector
• Vector – a DNA molecule (usually a bacterial
plasmid) that is specifically designed to carry
foreign DNA
– Selectable marker
– MCS
3. Restriction Enzymes
• Restriction Enzymes – cut DNA at particular
sequences
– Ex: EcoR1 – recognizes GAATTC
– 100’s of RE’s
Transformation
• Get “competent” bacteria
(usually E.coli that is
capable of being
transformed)
• Treat those cells to
calcium chloride
• Heat shock to force
bacterial cell to take up
plasmid DNA
Now…
• Select for bacteria
that have taken up
your plasmid
(usually use
antibiotic)
• Grow bacteria in
large quantities,
purify out protein,
and…
PCR (polymerase chain reaction)
• Specific sequence of DNA is amplified (copied
many times)
• Requires:
– DNA template (contains your gene of interest)
– Tac polymerase (a DNA polymerase that can work
at high temperatures)
– Nucleotides (to synthesize new DNA)
– Primers (specific to the gene of interest)
• PCR is a cyclic
process:
– Heat to denature
(open up) DNA
– Cool to allow primers
to anneal (stick to)
single stranded DNA
– Warm to allow DNA
to be replicated by
polymerase
– REPEAT
Gel Electrophoresis
• Uses a gel as a molecular sieve to separate
pieces of DNA based on size
• Smaller pieces travel further than larger pieces
Let’s discuss some examples
1. Genetically modified food
2. Transgenic animals
3. Pharmaceuticals
1. Genetically Modified Food
• Insert particular genes
within a type of food to
make it better tasting,
longer lasting, or more
healthy.
• Long term effects of
consumption of this
food are unknown.
2. Transgenic Animals
• Research purposes
– Knock outs
– Overexpression
– Use of GFP to select for
animals that are transgenic
• Consumer purposes (food
or medication)
3. Pharmaceuticals
• Use plants or other GMO to produce pharmaceuticals
– Ex: Gaucher’s disease (lysosomal storage disease – buildup
of fat in cells); Pfizer received patent to grow drug to treat
this disease in tobacco plant
– Ex: Use of a transgenic goat to produce an anticoagulant (in
the goat milk) to reduce probability of blood clots
• Use bacteria to produce insulin, human growth
hormone, clotting factors for hemophaelics
• GMO mammals (mice) used in pharmaceutical
research labs to study disease and treatment
Your Homework:
• Read the entire laboratory handout. You must
carefully read the background information AND
the laboratory procedure as well.
• Answer the pre-lab questions.