Detecting single molecules and sequencing DNA

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Detecting single molecules and sequencing DNA

  1. 1. Detecting single molecules and sequencing DNA Rohan T. Ranasinghe University Chemical Laboratories, Lensfield Road, Cambridge CB2 1EW
  2. 2. Locations and timeline 1 mile
  3. 3. Locations and timeline 1 mile http://www.cambridge 2000.com Old Cavendish Laboratory 1953: Discovery of the structure of DNA
  4. 4. Locations and timeline 1 mile http://www.cambridge 2000.com Old Cavendish Laboratory 1953: Discovery of the structure of DNA LMB 1977: Sanger method for sequencing invented http://www2.mrc-lmb.cam.ac.uk
  5. 5. Locations and timeline 1 mile http://www.cambridge 2000.com Old Cavendish Laboratory 1953: Discovery of the structure of DNA Sanger Institute 1993: Work on Human genome project at the Sanger starts Genome Research Ltd. LMB 1977: Sanger method for sequencing invented http://www2.mrc-lmb.cam.ac.uk
  6. 6. Locations and timeline 1 mile http://www.cambridge 2000.com Old Cavendish Laboratory 1953: Discovery of the structure of DNA Chemistry department http://www.flickr.com/ photos/shai- bl/5584629687/sizes/ m/in/photostream/ 1997: Work on Solexa method for sequencing started Sanger Institute 1993: Work on Human genome project at the Sanger starts Genome Research Ltd. LMB 1977: Sanger method for sequencing invented http://www2.mrc-lmb.cam.ac.uk
  7. 7. Structure of DNA http://www.themicrobiologist.com Solved in Cambridge in 1953 by James Watson and Francis Crick using data collected by Rosalind Franklin and Maurice Wilkins at King’s College London The key to the structure was base pairing
  8. 8. Structure of DNA http://www.themicrobiologist.com Solved in Cambridge in 1953 by James Watson and Francis Crick using data collected by Rosalind Franklin and Maurice Wilkins at King’s College London The key to the structure was base pairing
  9. 9. Structure of DNA http://www.flickr.com/photos/grahams__flickr /504365411/sizes/l/in/photostream/ Solved in Cambridge in 1953 by James Watson and Francis Crick using data collected by Rosalind Franklin and Maurice Wilkins at King’s College London The key to the structure was base pairing
  10. 10. Structure of DNA http://www.flickr.com/photos/major_clanger/ 5881631482/sizes/o/in/photostream/ http://www.flickr.com/photos/grahams__flickr /504365411/sizes/l/in/photostream/ Solved in Cambridge in 1953 by James Watson and Francis Crick using data collected by Rosalind Franklin and Maurice Wilkins at King’s College London The key to the structure was base pairing
  11. 11. Structure of DNA http://www.flickr.com/photos/major_clanger/ 5881631482/sizes/o/in/photostream/ http://www.flickr.com/photos/grahams__flickr /504365411/sizes/l/in/photostream/ Solved in Cambridge in 1953 by James Watson and Francis Crick using data collected by Rosalind Franklin and Maurice Wilkins at King’s College London The key to the structure was base pairing The fidelity of the Watson-Crick base pairs and the double helix structure are the cornerstones of DNA sequencing and modern forensic science
  12. 12. DNA Sequencing Why would you want to sequence DNA? http://www.sikeston.k12.mo.us
  13. 13. DNA Sequencing Why would you want to sequence DNA? http://www.sikeston.k12.mo.us © Invitrogen
  14. 14. DNA Sequencing Why would you want to sequence DNA? A genome contains the information required to build an organism http://www.sikeston.k12.mo.us © Invitrogen
  15. 15. DNA Sequencing Why would you want to sequence DNA? A genome contains the information required to build an organism http://www.sikeston.k12.mo.us It’s a long book... © InvitrogenWikipedia
  16. 16. DNA Sequencing Why would you want to sequence DNA? A genome contains the information required to build an organism http://www.sikeston.k12.mo.us It’s a long book... © Invitrogen ~3,000,000,000 (3 ×109) letters in each of the ~1014 cells in a human Wikipedia
  17. 17. DNA Sequencing Why would you want to sequence DNA? A genome contains the information required to build an organism http://www.sikeston.k12.mo.us It’s a long book... © Invitrogen ~3,000,000,000 (3 ×109) letters in each of the ~1014 cells in a human Distance between base pairs = 0.34 nm (0.34 ×10-9 m) Wikipedia
  18. 18. DNA Sequencing Why would you want to sequence DNA? A genome contains the information required to build an organism http://www.sikeston.k12.mo.us It’s a long book... © Invitrogen ~3,000,000,000 (3 ×109) letters in each of the ~1014 cells in a human The DNA in one of your cells would be 2 m long in the B-form structure Distance between base pairs = 0.34 nm (0.34 ×10-9 m) Wikipedia
  19. 19. T Sanger sequencing CAGTCAGTCA GA C G T A C G TA C G T AC Based on copying of DNA: Genome Research Ltd.
  20. 20. Sanger sequencing CAGTCAGTCA GA C G A C TA G T C Based on copying of DNA: Genome Research Ltd.
  21. 21. Sanger sequencing CAGTCAGTCA GA C G T A C TA G T C Based on copying of DNA: Genome Research Ltd.
  22. 22. Sanger sequencing CAGTCAGTCA GA C G T A C TA CG T C Based on copying of DNA: Genome Research Ltd.
  23. 23. Sanger sequencing CAGTCAGTCA GA C G T A C TA CG T A C Based on copying of DNA: Genome Research Ltd.
  24. 24. Sanger sequencing CAGTCAGTCA GA C G T A C G TA CG T A C Based on copying of DNA: Genome Research Ltd.
  25. 25. T Sanger sequencing CAGTCAGTCA GA C G T A C G TA CG T A C Based on copying of DNA: Genome Research Ltd. Incorporation of fluorescent nucleotide terminates the copying process
  26. 26. T Sanger sequencing CAGTCAGTCA GA C G T A C G TA CG T A C Based on copying of DNA: Repeat ~1030 times Genome Research Ltd.
  27. 27. T Sanger sequencing CAGTCAGTCA GA C G T A C G TA CG T A C Based on copying of DNA: Repeat ~1030 times Genome Research Ltd.
  28. 28. Sanger sequencing Copied sequence G C T A C G A T G C T A C G A T G C T A Original sequence Repeat 3 × 108 times to read genome (would take another 190 years at this speed!*) *Note: original animation took ~20 seconds)
  29. 29. The human genome project http://www.c-spanvideo.org/program/157909-1 Started: 1989 (in the USA)
  30. 30. The human genome project First draft completed: 2000 ‘Finished’: 2003 http://www.c-spanvideo.org/program/157909-1 Started: 1989 (in the USA)
  31. 31. The human genome project First draft completed: 2000 ‘Finished’: 2003 http://www.c-spanvideo.org/program/157909-1 Started: 1989 (in the USA)
  32. 32. The human genome project Cost: $3,000,000,000 First draft completed: 2000 ‘Finished’: 2003 http://www.c-spanvideo.org/program/157909-1 Started: 1989 (in the USA)
  33. 33. The human genome project Cost: $3,000,000,000 First draft completed: 2000 ‘Finished’: 2003 http://www.flickr.com/photos/93425126@N00/43948 34217/in/set-72157623515077498/ http://www.c-spanvideo.org/program/157909-1 Started: 1989 (in the USA)
  34. 34. The human genome project Cost: $3,000,000,000 First draft completed: 2000 ‘Finished’: 2003 http://www.flickr.com/photos/93425126@N00/43948 34217/in/set-72157623515077498/ http://www.c-spanvideo.org/program/157909-1 Started: 1989 (in the USA) UK effort on the Human Genome Project largely carried out in this building in the Sanger Centre
  35. 35. The human genome project Cost: $3,000,000,000 First draft completed: 2000 ‘Finished’: 2003 http://www.flickr.com/photos/93425126@N00/43948 34217/in/set-72157623515077498/ http://www.c-spanvideo.org/program/157909-1 Started: 1989 (in the USA) UK effort on the Human Genome Project largely carried out in this building in the Sanger Centre 9 Chromosomes were sequenced here (about a third of the genome)
  36. 36. What does it mean to detect a single molecule? Looking for a needle in a haystack?
  37. 37. What does it mean to detect a single molecule? Looking for a needle in a haystack? How many blades of grass on a football pitch?
  38. 38. What does it mean to detect a single molecule? Looking for a needle in a haystack? About 200,000,000 or 2×108 How many blades of grass on a football pitch?
  39. 39. What does it mean to detect a single molecule? How many molecules in a vial of water? Looking for a needle in a haystack? About 200,000,000 or 2×108 How many blades of grass on a football pitch?
  40. 40. What does it mean to detect a single molecule? 18 mL (1 mole) of water contains Avogadro’s number of molecules: 6.02 ×1023 How many molecules in a vial of water? Looking for a needle in a haystack? About 200,000,000 or 2×108 How many blades of grass on a football pitch?
  41. 41. What does it mean to detect a single molecule? 18 mL (1 mole) of water contains Avogadro’s number of molecules: 6.02 ×1023 How many molecules in a vial of water? Looking for a needle in a haystack? About 200,000,000 or 2×108 How many blades of grass on a football pitch? So 1 mole of grass blades would cover 6.02 ×1023 ÷ 2×108 = 3 ×1015 football pitches
  42. 42. What does it mean to detect a single molecule? 18 mL (1 mole) of water contains Avogadro’s number of molecules: 6.02 ×1023 How many molecules in a vial of water? Looking for a needle in a haystack? About 200,000,000 or 2×108 How many blades of grass on a football pitch? So 1 mole of grass blades would cover 6.02 ×1023 ÷ 2×108 = 3 ×1015 football pitches That’s a lot of haystacks...
  43. 43. What does it mean to detect a single molecule? 1 mole of grass blades = 3×1015 football pitches = 15×1012 km2
  44. 44. What does it mean to detect a single molecule? 1 mole of grass blades = 3×1015 football pitches = 15×1012 km2 Surface area of Earth = 5×108 km2 (1011 football pitches!)
  45. 45. What does it mean to detect a single molecule? 1 mole of grass blades = 3×1015 football pitches = 15×1012 km2 Surface area of Jupiter = 6×1010 km2 *Lab demonstration: 180 µL (15×1010 km2 of grass blades)
  46. 46. What does it mean to detect a single molecule? 1 mole of grass blades = 3×1015 football pitches = 15×1012 km2 Surface area of the Sun = 6×1012 km2
  47. 47. What does it mean to detect a single molecule? 1 mole of grass blades = 3×1015 football pitches = 15×1012 km2 Surface area of the Sun = 6×1012 km2 1 mole of grass blades would cover the surface area of about 2.5 Suns!
  48. 48. What does it mean to detect a single molecule? 1 mole of grass blades = 3×1015 football pitches = 15×1012 km2 Surface area of the Sun = 6×1012 km2 1 mole of grass blades would cover the surface area of about 2.5 Suns! All images: nasa.gov
  49. 49. Sanger sequencing Sanger sequencing uses about 2×1010 molecules per 100 letters
  50. 50. Solexa sequencing Invented in 1997 in this department Developed by a spin-out company in Saffron Walden Sold for $650,000,000 in 2006
  51. 51. Solexa sequencing Solexa sequencing uses about 103 molecules to read 100 letters About as many blades of grass as on the penalty spot Imaging technology: lab demonstration http://thesportboys.wordpress.com/category/international/page/2/ Invented in 1997 in this department Developed by a spin-out company in Saffron Walden Sold for $650,000,000 in 2006
  52. 52. Solexa sequencing
  53. 53. Solexa sequencing © Royal Society of Chemistry publishing Densely packed microscopic “islands” of DNA generate information very quickly
  54. 54. Solexa sequencing © Royal Society of Chemistry publishing • “Recycled” template molecules ready for a incorporation of the next fluorescent letter • Possible to read about 100 letters from each DNA strand, rather than 1
  55. 55. Solexa sequencing © Royal Society of Chemistry publishing Genome Research Ltd.
  56. 56. Solexa sequencing © Royal Society of Chemistry publishing Genome Research Ltd. Cost to sequence a human genome: around $10,000 Time to sequence a human genome: less than a week First African, Asian and giant panda genomes sequenced Sanger Institute owns 37 instruments
  57. 57. Solexa sequencing © Royal Society of Chemistry publishing Genome Research Ltd. Cost to sequence a human genome: around $10,000 Time to sequence a human genome: less than a week First African, Asian and giant panda genomes sequenced Sanger Institute owns 37 instruments
  58. 58. Summary The structure of DNA, discovered in 1953 has been crucial to sequencing the human genome The first human genome was sequenced using Fred Sanger’s method, invented in 1977. The project ran for 14 years, costing $3 billion New methods for sequencing use single molecule detection to dramatically accelerate the decoding process One approach using single molecule techniques, invented by Shankar Balasubramanian and David Klenerman in our department in 1997 is now widely used for sequencing worldwide The cost of sequencing has fallen to $10,000 and takes less than a week

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