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: 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 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.