BILD Notes

BILD Notes - BILD 10: 11/03 Notes: Applications to Genetic...

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BILD 10: 11/03 Notes: Applications to Genetic Engineering - Reduce production cost of specific proteins - Produce improved crops and livestock - Forensic and paternity cases - Determine function of a gene - Determine cause of disease - Design improved drugs - Gene therapy Prof. Green is old Prof. Green is wearing a shirt that says Caffeine How do restriction enzymes work? - Restriction enzymes cut palindromic sequences - i.e ===GAATTC==========GAATTC=== ===CTTAAG==========CTTAAG=== - Restriction enzyme cuts the DNA into fragments. - MADAM - is a palindrome. - Palindrome is a something that can be spelled the same backwards and forwards. * Note - I did not know that. - Addition of a DNA fragment from another source; the two fragments stick together by base pairing. - AGC CGA Then fill in the base pairs. - AGC G C T T C G CGA - Restriction enzymes have two active sites -- one to cut Watson strand, and one to cut the Crick strand. - Palindromic sequences are usually 6 pairs - After the enzymes cut the DNA, it begins to fall apart. - Why is that important? Genetic engineers recognize this because the DNA strands are palindromic and sticky therefore, allowing it to be joined together from two different sources - DNA ligase seals the strands. - The new strand is called Recombinant DNA Gel electrophoresis of DNA - Some weird machine - Uses gel and forms bands depending on the size of the fragment. - The bigger the fragment of DNA, the harder it is to move. - Find them on the gel by two techniques. 1. Staining of florescent dye. Use UV light and you will be able to see it. 2. Make your DNA radioactive. - Using both ways, you will be able to see your DNA as bands. Common Cloning Vectors
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- Cloning Vector: An agent used to introduce new DNA into a host cell. - Plasmids: Small circular DNA molecules found in many bacteria. - Phage: viruses that specifically infect bacteria; some leave many surviving cells with viral DNA integrated into host cell's DNA. - Mammalian Viruses: Use defective viruses that don't kill infected host cells, but integrate into host's DNA. - Cosmid: combination of a small plasmid with a lambda cos restriction site, useful in transferring long DNA segments. Synopsis of Gene Cloning - Ex: Human gene for insulin. - Cut plasmid with restriction enzyme (R.E.) - Cut human DNA with same R.E. - Join cut plasmid to human DNA fragments with DNA ligase to create recombinant DNA plasmids. - Transfect E. Coli with recombinant DNA plasmids - Detect E. Coli colonies (clones) that produce human insulin - These clones produce lots of insulin because every cell has many copies (50-100) of the insulin gene. - E. Coli look like rods. - They look nasty - Genintech - a San Francisco area bioengineering company. - Kelly didn't bring cereal today.
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BILD Notes - BILD 10: 11/03 Notes: Applications to Genetic...

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