TECHNIQUES IN BIOTECHNOLOGY

1. TECHNIQUES IN BIOTECHNOLOGY

2. DNA EXTRACTION • http://learn.genetics.utah.edu/content/labs/extraction/

3. Purpose: to purify a DNA sample • Uses • genetic testing • crime scene investigation • gene studies

4. Steps: • collect cells • remove DNA from cell • purify it • use it!

5. Ex. Insert for plasmid • From someone else’s DNA • ex. fish gene in strawberries, • jellyfish gene in plants

6. In order to do these things, we need a way to make many copies of the genes we want

7. Making more DNA: PCR • purpose: to make many copies of a specific piece of DNA Steps: • denature DNA using temperature • add in the ingredients (ATGC, enzymes, primers) to copy it • repeat

9. Making an insert: Polymerase Chain Reaction

11. Uses • make the desired gene/piece of DNA to use it to make proteins, study it, etc.

12. To Make a Recombinant Plasmid: Insert • Cut the plasmid and the insert with the same restriction endonuclease to make complementary sticky ends. • Combine the sticky ends using ligase. • ligase: enzyme used to join DNA together 3. Introduce the recombinant plasmid into bacteria.

13. Making a Recombinant Plasmid

15. - - - - - - - - + - + - - - - ++ -++ - - ++ - ++ + + + + + - - - - - - - - - - - - + + + + + Bacterial Transformation • introduction of foreign DNA into a bacterial cell • plasmid is used as a vector, a vehicle by which DNA can be introduced into host cell phospholipid bilayer Ca2+ ions plasmid

16. Following transformation bacteria are grown in medium with antibiotic… Only the bacteria that have the plasmid (and therefore the antibiotic resistance) will survive.

17. Example plasmid: Origin of Replication: • where the plasmid starts to duplicate itself • the specific sequence MUST NOT be cut by restriction endonucleases or it won’t be able to replicate

18. Using Bacteria as Production Factories • easy to grow • no ethical issues • small genome • easy to manipulate

19. DNA SEQUENCING • purpose: find out the order of DNA, the “fingerprint” • uses: forensics, genetic testing, studying mutations

24. Steps • like PCR, put your template (DNA you want to “read”) with the nucleotides, primers, enzymes, and use heat to make “more” • some of the nucleotides have fluorescent labels and stop the chain from being made

25. results in a soup of different-length fragments • separate them by size, and “read”

26. Gel Electrophoresis • used to “see” the DNA • used in sequencing and to determine the origin of DNA ex. Tsunami baby

27. Sequencing

28. Ex. RFLP: Restriction Fragment Length Polymorphism Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals

29. Steps

30. CLONING http://learn.genetics.utah.edu/content/tech/cloning/

31. Therapeutic cloning • used to produce tissue that is identical to the donor, to prevent rejection

32. Reproductive Cloning • creates an organism with the same genetic material (DNA) as the original organism – an EXACT COPY of the donor

34. Dolly the Sheep • the first cloned sheep

35. Common uses of biotechnology: • Making "stuff” • proteins, enzymes, medication, etc. can be produced by engineered bacteria! • Food can be altered to have new traits • Cloning (therapeutic and reproductive) • Genetic screening • crime cases, relationship, genetic screening, etc. 3. Gene Therapy

36. Gene therapy • desired gene is inserted into cell's nucleus using a retrovirus as a carrier • defective gene replaced by functional gene

37. Ex. ADA deficiency • adenosine deaminase deficiency • little immunity with low chances of recovery - the T-cells of a four-year-old were removed, modified and re-inserted to fix her immune system