Biotechnology Recombinant DNA

1. Biotechnology & Recombinant DNA

2. Recombinant DNA

3. What is biotechnology? Using living microorganisms or cell components to make products Often via genetic engineering and using recombinant DNA

4. What is recombinant DNA? Recall: happens naturally Transposons Researchers can also produce it Use bacterial plasmids or viruses (bacteriophages) Uses Bacteria make insulin yeast help make components of Hepatitis B vaccine

5. How is recombinant DNA made? Inserting foreign DNA into a bacterial cell Use restriction enzymes, plasmids, ligase and bacterial host Restriction enzymes Defense against viruses Clone gene or make gene product

6. What are restriction enzymes? Naturally occur in bacteria Bacteria use these to combat viral infection Bacteria DNA uneffected because some is methylated Sequence-specific cloning animation

7. What is a vector? Different from a disease vector! plasmid animation Plasmid or virus Used to insert DNA into host cell Must be able to self-replicate! Must be small so not fragile Both types of vectors can allow researchers to clone DNA But there�s another approach to DNA amplification�

8. PCR: making copies of DNA

9. What is PCR? Stands for _____________ Need primers of about 20 bp to start DNA polymerase doesn�t start reaction, only lengthens Primers recognize regions which flank target gene Ingredients primers DNA polymerase from TAC (Thermus aquaticus) Individual nucleotides Can you go over that one more time? �PCR animation

10. Why would you use PCR? Detect small amounts of DNA Can you think of examples? Get in groups and discuss! Forensics Infectious agents Gene mapping Human Genome Project Taxonomy and systematics studies Cancer and study of other human diseases Sequencing of rRNA and mRNA via cDNA

11. Let�s take a brief look at some of these�

12. DNA fingerprinting

13. What is gel electrophoresis? Sorting DNA segments by size Process is called Southern Blotting

14. What is Southern blotting? After gel electrophoresis Filter paper blots DNA off Radioactive probes added Autoradiography pinpoints sequence Southern Blot animation

15. When would you use gel electrophoresis? DNA fingerprinting Restriction enzymes create restriction fragment length polymorphisms (RFLPs)

16. What does DNA fingerprinting look like?

17. Is this the only way to �look� at DNA? No�we can sequence it, too! Sanger sequencing animation

18. Cloning

19. How do researchers get the DNA they want to clone? Gene libraries Either plasmid or phage libaries Fine for prokaryotic DNA Problem with eukaryotic DNA�

20. So what�s the problem? Eukaryotic DNA has introns� Must make complementary DNA (cDNA) Use reverse trasncriptase cDNA animation Now DNA can be inserted�

21. What do they do with the cloned DNA? Lots of different things!

22. What are bioreactors? Using bacteria to produce gene products Insulin: diabetes Human growth hormone Cellulase (break down cell wall for animal feed) Factor VIII: hemophilia

23. What are plant GMOs? Genetically modified organisms Transgenic plant or animal Bioreactors filled with these Plants Cotton, corn, potato to make pest resistant Soybeans resistant to common herbicide Some corn, cotton are herbicide and pest resistant Could produce human hormones, clotting factors, antibodies on seeds in future

24. What are animal GMOs? Foreign genes into embryos produce animals that manufacture human hormones, etc. = gene pharming Blood clotting factor goats Sheep milk with human alpha-1-antitrypsin (used to treat heritable emphysema) 1997, Dolly Since then, cloned sheep, cows, goats, mice Humans: moratorium

25. What is gene therapy? Insertion of genetic material into human cells to treat a disorder Use retrovirus to insert normal gene into cell Healthy genes to make up for faulty genes Severe combined immunodeficiency syndrome 1990, girl received normal gene in white blood cells Using genes to treat other illnesses