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Ms. He DNA technology


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Ms. He DNA technology

  1. 1. BIOTECHNOLOGYWhat can we do with DNA?
  2. 2. Biotechnology• Manipulation of biological organisms or their components for research and industrial purpose• Usually manipulate DNA itself
  3. 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. 4. Basic techniques1. DNA isolation2. Restriction enzyme digest3. DNA amplification • Transformation and growth – In vivo • Polymerase Chain Reaction (PCR) – In vitro4. Gel electrophoresis
  5. 5. DNA IsolationBefore DNA can be manipulated, it needs to be isolatedfrom the cells.1.Disrupt cell membranes with a detergent • Example of detergent: SDS, Tween-202.Precipitate DNA with ethanol3.Obtain precipitated DNA and storage
  6. 6. DNA Isolation
  7. 7. DNA isolation How do you get specific sequence from the entire genome?
  8. 8. Restriction Enzyme Digestion• DNA must be cut into smaller pieces before they can be used in other techniques.
  9. 9. Restriction Enzymes• Molecular scissors• Restriction Endonucleases: digestive enzymes that recognize specific DNA sequences (known as Recognition site) and cut at specific points
  10. 10. Restriction Site• Typically 4-8 bp in length• Double-stranded DNA• Always palindromic: 5’ G A A T T C 3’ 3’ C T T A A G 5’ EcoRI recognition site What does palindromic mean? Same sequence on complementary strand in opposite orientation
  11. 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. 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. 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. 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. 15. blunt ends – enzyme digests to make straight endsSmaI 5’ 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. 16. DNA LigaseT4 DNA ligase – used to chemically join sticky ends of DNA together
  17. 17. Recombinant DNAComplementary sticky ends from different pieces of DNA can be joined together – recombinant DNA
  18. 18. Restriction Enzyme Animation•• Tutotial:•
  19. 19. DNA AmplificationTransformation & growthPCR
  20. 20. Amplification of DNA (in vivo)Transformation & Growth What is transformation?• Treat bacteria to make cell walls permeable to uptake of foreign DNA• Transformed bacterial cell grow and divide to amplify DNA
  21. 21. Amplification of DNA (in vitro)PCR PCR = Polymerase Chain Reaction•Powerful technique to produce millions of copies ofspecific DNA. _________________________________________ _________________________________________
  22. 22. Successful PCR reaction1) Need something to replicate and someplace to start1) Need something to open DNA and unwind it • AND something to stabilize it once unwound1) Need something to provide the primer to initiate synthesis2) Need something to synthesize the new DNA
  23. 23. • 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
  24. 24. Successful PCR reaction1) Need something to replicate and someplace to start Template DNA You can chose (chromosome, plasmid, etc)1) Need something to open DNA and unwind it • AND something to stabilize it once unwound Heat1) Need something to provide the primer to initiate synthesis You can chose the primers, and therefore specify EXACTLY what you want to amplify2) Need something to synthesize the new DNA DNA polymerase (Must be heat stable) *Taq polymerase
  25. 25. Taq polymerase• Isolated from Thermophilus aquaticus bacterium• These bacteria live in hot springs and has heat stable enzyme that can withstand extreme temperatures
  26. 26. 3 steps for each PCR cycle1. DNA strand denaturation (95 oC) • Separate double strand DNA • Each strand becomes template strand2. Primer annealing (50 – 65 oC) • Short DNA pieces bind to temperate strands3. DNA strand synthesis (72 oC) • Produce new DNA strands
  27. 27. 2 copies of targetedsequence after 3rdcycle
  28. 28. Power of PCR• After 30 cycles, 230 (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
  29. 29. PCR animation•• 3D animation:•
  30. 30. Gel ElectrophoresisSeparating DNA sequence
  31. 31. Gel Electrophoresis• Separate DNA through a gel medium using an electric currentWhy can we move DNA with electricity?• DNA has a negative charge. Therefore it will move toward positive electrode
  32. 32. Gel MediumThe 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.
  33. 33. Liquid solutions of the gel is poured into a mould andallowed to set and solidify.
  34. 34. Loading DyeDNA is colourless.How do you know that it gets into the gel?• Coloured dyes are mixed with DNA to track distance travelled – Loading Dye
  35. 35. positive negativeelectrode electrode agarose gel
  36. 36. Separation DNA by sizeThe 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
  37. 37. Visualizing GelDNA 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
  38. 38. Ethidium Bromide