1. BIOTECHNOLOGY What can we do with DNA?

2. Biotechnology Manipulation of biological organisms or their components for research and industrial purpose Usually manipulate DNA itself

3. How to study individual gene? To study the function of individual genes, molecular biologists will cut them out of a genome and place them into bacteria Why study gene in bacteria?

4. Basic techniques 1. DNA isolation 2. Restriction enzyme digest 3. DNA amplification Transformation and growth – In vivo Polymerase Chain Reaction (PCR) – In vitro 4. Gel electrophoresis

5. DNA Isolation Before DNA can be manipulated, it needs to be isolated from the cells. 1. Disrupt cell membranes with a detergent Example of detergent: SDS, Tween-20 2. Precipitate DNA with ethanol 3. Obtain precipitated DNA and storage

6. DNA Isolation

7. DNA isolation How do you get specific sequence from the entire genome?

8. Restriction Enzyme Digestion DNA must be cut into smaller pieces before they can be used in other techniques.

9. Restriction Enzymes Molecular scissors Restriction Endonucleases: digestive enzymes that recognize specific DNA sequences (known as Recognition site) and cut at specific points

10. Restriction Site Typically 4-8 bp in length Double-stranded DNA Always palindromic: What does palindromic mean? 5’ G A A T T C 3’ 3’ C T T A A G 5’ EcoRI recognition site Same sequence on complementary strand in opposite orientation

11. Why use these enzyme? Restriction enzymes are naturally found in bacteria Restriction enzymes act as “immune system” of bacteria Protect bacteria against DNA from other organisms (ex. bacteriophage (bacterial virus)) Recognize and cut phosphodiester bonds of foreign DNA, not its own genome -> making foreign DNA harmless for the cell

12. Restriction enzyme digestion Restriction enzyme recognize a palindromic DNA sequence in double-stranded DNA and cleave both strands Resulting Sticky End: a single-stranded overhangs Sticky ends with 5’ overhang Sticky ends with 3’ overhang Blunt ends

13. EcoRI digestion 5’ G A A T T C 3’ 3’ C T T A A G 5’ 5’ G 3’5’ A A T T C 3’ 3’ C T T A A 5’ 3’ G 5’ 5’ overhang

14. PstI Digestion 5’ C T G C A G 3’ 3’ G A C G T C 5’ 5’ C T G C A 3’ 5’ G 3’ 3’ G 5’ 3’ A C G T C 5’ 3’ overhang

15. blunt ends – enzyme digests to make straight ends SmaI5’ C C C G G G 3’ 3’ G G G C C C 5’ 5’ C C C 3’5’ G G G 3’ 3’ G G G 5’3’ C C C 5’

16. DNA Ligase T4 DNA ligase – used to chemically join sticky ends of DNA together

17. Recombinant DNA Complementary sticky ends from different pieces of DNA can be joined together – recombinant DNA

19. Restriction Enzyme Animation http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf Tutotial: http://www.dnalc.org/resources/animations/restriction.html http://www.dnalc.org/resources/animations/restriction.html

20. DNA AMPLIFICATION Transformation & growth PCR

21. Amplification of DNA (in vivo) Transformation & Growth Treat bacteria to make cell walls permeable to uptake of foreign DNA Transformed bacterial cell grow and divide to amplify DNA What is transformation?

22. Amplification of DNA (in vitro) PCR PCR = Polymerase Chain Reaction Powerful technique to produce millions of copies of specific DNA. _________________________________________

23. Successful PCR reaction 1) Need something to replicate and someplace to start 1) Need something to open DNA and unwind it AND something to stabilize it once unwound 1) Need something to provide the primer to initiate synthesis 2) Need something to synthesize the new DNA

24. What you need: Template DNA dNTPs – nucleotides (dATP, dTTP, dCTP, dGTP) Two specific Primers: short pieces (20-30 nucleotides) of synthetic single-stranded DNA First is complementary to one DNA strand at the beginning of the target region Second is complementary to opposite DNA stand at the end of the targeted region DNA polymerase –Taq polymerase

25. Successful PCR reaction 1) Need something to replicate and someplace to start 1) Need something to open DNA and unwind it AND something to stabilize it once unwound 1) Need something to provide the primer to initiate synthesis 2) Need something to synthesize the new DNA Template DNA (chromosome, plasmid, etc) You can chose Heat You can chose the primers, and therefore specify EXACTLY what you want to amplify DNA polymerase (Must be heat stable) *Taq polymerase

26. Taq polymerase Isolated from Thermophilus aquaticus bacterium These bacteria live in hot springs and has heat stable enzyme that can withstand extreme temperatures

27. 3 steps for each PCR cycle 1. DNA strand denaturation (95 o C) Separate double strand DNA Each strand becomes template strand 2. Primer annealing (50 – 65 o C) Short DNA pieces bind to temperate strands 3. DNA strand synthesis (72 o C) Produce new DNA strands

28. * * 2 copies of targeted sequence after 3 rd cycle

29. Power of PCR After 30 cycles, 2 30 (more than a billion) copies of DNA can be produced 30 cycles of PCR takes ~1-2 hours to complete - PCR is preformed using thermal cycler

30. PCR animation http://www.sumanasinc.com/webcontent/animations/conte nt/pcr.html http://www.sumanasinc.com/webcontent/animations/conte nt/pcr.html 3D animation: http://www.dnalc.org/view/15475-The-cycles-of-the- polymerase-chain-reaction-PCR-3D-animation-with-no- audio.html http://www.dnalc.org/view/15475-The-cycles-of-the- polymerase-chain-reaction-PCR-3D-animation-with-no- audio.html

32. GEL ELECTROPHORESIS Separating DNA sequence

33. Gel Electrophoresis Separate DNA through a gel medium using an electric current Why can we move DNA with electricity? DNA has a negative charge. Therefore it will move toward positive electrode

35. Gel Medium The gel medium can be made from: 1. agarose - seaweed extract 2. polyacrylamide - artificial polymer The type of gel used is dependent on how well separated the DNA pieces need to be. Polyacrylamide has higher resolution than agarose.

36. Liquid solutions of the gel is poured into a mould and allowed to set and solidify.

37. Loading Dye DNA is colourless. How do you know that it gets into the gel? Coloured dyes are mixed with DNA to track distance travelled – Loading Dye

38. negative electrode positive electrode agarose gel

39. Separation DNA by size The gel provides resistance for DNA movement. Short DNA moves through gel easily travels further in a set amount of time Long DNA requires more effort to move through gel does not move as far in a set amount of time

42. Visualizing Gel DNA is colourless. How do you see where the DNA is after the separation is complete? DNA is stained with ethidium bromide UV light box is used to see fluorescent DNA bands

44. Ethidium Bromide