Agenda (Monday-Wednesday)

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1 Agenda (Monday-Wednesday) Chapter 12 Recombinant DNA Technology Recombinant DNA Techniques DNA Fingerprinting and Forensic Science DNA Fingerprinting Techniques Pre-lab 8 activities Tomorrow: Day One of Lab 8 Lecture: PCR Wednesday: Day Two of Lab 8 Lecture: Biology of Viruses What is Recombinant DNA technology? Lab techniques for combining genes from different sources into a single DNA molecule How and why do scientists use these tools? One trick is to created transgenic organisms Such as a bacterial cell that can generate human proteins! Unsuspecting Bacterium From a human cell, snip out the gene for insulin Insert human insulin GENE into bacterial genome Allow bacteria to grow, replicate and transcribe and translate insulin! From bacteria, purify out human insulin. Sell for billions of dollars $$! How about a transgenic mammal? Drug Animal Value/Animal/Yr* AAT sheep $15,000 tpa goat 75,000 Factor VIII sheep 37,000 Factor IX sheep 20,000 Hemoglobin pig 3,000 Lactoferrin cow 20,000 CFTR sheep, mouse 75,000 Human Protein C pig 1,000,000 How can this possibly be done, you ask? Well, molecular biologists have cool tools DNA Scissors = Restriction Enzymes DNA Glue = Ligase 1

2 Restriction Enzymes Restriction Enzymes Restriction Enzymes These are bacterial enzymes The biological function of these enzymes is to protect bacterial cells from virus invasion!! If viral DNA enters a bacterial cell, restriction enzymes (endonucleases) will snip them up! Viruses infecting a bacterium EcoR1 EcoR1 was one of the first restriction enzymes purified. (From E. Coli) EcoR1 EcoR1 was one of the first restriction enzymes purified. (From E. Coli) EcoR1 recognizes the sequence: GAATTC CTTAAC EcoR1 recognizes the sequence: GAATTC CTTAAC This is HOW EcoR1 cuts this sequence! Bacterial Cell genome Human cell, with human gene for insulin human gene for insulin cut out of human chromosome, and glued into bacterial chromosome Bacterial cell making protein of interest! 2

3 DNA Fingerprinting Used in forensics, paternity suits, victim identification, evolutionary biology How could we utilize restriction enzymes to tell one person from another? In a crime lab, DNA fingerprinting can be used to tell one person from another HOW??? How do we figure out the LENGTHS of the DNA generated by our digests? What is (are) the length(s) of the DNA sitting in a microtube? DNA agarose gels Agarose is a little like jello Forms a semi-solid gel matrix Different DNA samples added to different wells of the gelmatrix Gel is placed in an electric field DNA moves in response to the electric field (DNA is a charged molecule) Electrophoresis - electrode wells Larger Electrophoresis Electrophoresis is how we sort the pieces out, by decreasing size. Sorting these out DNA 50 bp; 150 bp; 100 bp electrode Smaller 3

4 Electrophoresis Electrophoresis takes advantage of the negative charge DNA has. (The phosphate group has a negative charge). DNA, has an overall, negative charge. DNA, therefore, will be attracted to a positive electrode. We can place the DNA sample into a well of an agarose gel. The gel is subjected to an electrical field. The DNA will migrate towards the positive end of the box, through the agarose gel. DNA DNA: digest samples loading dye Larger /molecules remain towards the top, while smaller /molecules migrate further, faster! 4

5 MOVIE: DNA GEL WE NORMALLY INCLUDE A LADDER ON OUR AGAROSE GELS. A LADDER CONSISTS OF MULTIPLE DNA FRAGMENTS OF KNOWN SIZES USE THE LADDER TO ANALYZE THE LENGTH OF THE DNA FRAGMENTS IN THE OTHER LANES!!! er/animations/chrom%26elpho.html Staining and Destaining of Gels Place gel in fast-blast stain Transfer gel to water-filled weigh-boat. Shake gently Bands should begin to appear. Take 2 photos of your gel, use photo (or gel) to sketch a picture of your gel. MAKE SURE TO LABEL GEL PICTURE! 5