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 food2. Transgenic animals3. 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.