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Detecting and sequencing DNA at the single molecule level
- 1. Detecting single molecules and sequencing DNA Rohan T. Ranasinghe Director of Studies in Natural Sciences @RTRanasinghe https://www.slideshare.net/ ro26787/stoke-chemistry- masterclass
- 2. The other Director of Studies in Chemistry Available on: iTunes (https://goo.gl/hwMEFG) SoundCloud (https://goo.gl/s8p8Vd) YouTube (www.youtube.com/channel/UCWv6XjH8pzm WMVuE_pwz1mg) Twitter: @SteveTheChemist and @theevanslab.
- 3. Solexa sequencing • Invented in 1997 in the chemistry department by David Klenerman and Shankar Balasubramanian • Developed by a spin-out company in Saffron Walden
- 4. Solexa sequencing • Invented in 1997 in the chemistry department by David Klenerman and Shankar Balasubramanian • Developed by a spin-out company in Saffron Walden • Sold for £420,000,000 in 2006
- 5. Solexa sequencing • Invented in 1997 in the chemistry department by David Klenerman and Shankar Balasubramanian • Developed by a spin-out company in Saffron Walden The latest version of the DNA sequencer they invented (costs about £1,000,000) • Sold for £420,000,000 in 2006
- 6. Locations and timeline 1 mile
- 7. Locations and timeline 1 mile http://www.cambridge 2000.com Old Cavendish Laboratory 1953: Discovery of the structure of DNA
- 8. 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
- 9. 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
- 10. 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
- 11. 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
- 12. 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
- 13. 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
- 14. 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
- 15. 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
- 16. DNA Sequencing Why would you want to sequence DNA?
- 17. DNA Sequencing Why would you want to sequence DNA? A genome contains the information required to build an organism
- 18. DNA Sequencing Why would you want to sequence DNA? A genome contains the information required to build an organism It’s a long book... Wikipedia
- 19. DNA Sequencing Why would you want to sequence DNA? A genome contains the information required to build an organism It’s a long book... ~6,000,000,000 (6 ×109) letters in most of the ~4×1013 cells in a human Wikipedia
- 20. DNA Sequencing Why would you want to sequence DNA? A genome contains the information required to build an organism It’s a long book... ~6,000,000,000 (6 ×109) letters in most of the ~4×1013 cells in a human Distance between base pairs = 0.34 nm (0.34 ×10-9 m) Wikipedia
- 21. DNA Sequencing Why would you want to sequence DNA? A genome contains the information required to build an organism It’s a long book... ~6,000,000,000 (6 ×109) letters in most of the ~4×1013 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
- 22. T Sanger sequencing CAGTCAGTCA GA C G T A C G TA C G T AC Based on copying of DNA: Genome Research Ltd.
- 23. T Sanger sequencing CAGTCAGTCA GA C G T A C G TA CG T A C Based on copying of DNA: Genome Research Ltd.
- 24. T Sanger sequencing CAGTCAGTCA GA C G T A C G TA CG T A C Based on copying of DNA: Genome Research Ltd.
- 25. Chemistry of Sanger sequencing C C
- 26. Chemistry of Sanger sequencing deoxynucleotide triphosphate C C 3’-hydroxyl group
- 27. Chemistry of Sanger sequencing deoxynucleotide triphosphate Dye-labelled dideoxynucleotide triphosphate C C 3’-hydroxyl group
- 28. Chemistry of Sanger sequencing Copied strandTemplate strand
- 29. Chemistry of Sanger sequencing deoxynucleotide triphosphate Copied strandTemplate strand
- 30. Chemistry of Sanger sequencing deoxynucleotide triphosphate Copied strandTemplate strand
- 31. Chemistry of Sanger sequencing Copied strandTemplate strand
- 32. Chemistry of Sanger sequencing Copied strandTemplate strand
- 33. Chemistry of Sanger sequencing Copied strandTemplate strand
- 34. Chemistry of Sanger sequencing Copied strandTemplate strand
- 35. Chemistry of Sanger sequencing Copied strandTemplate strand
- 36. Chemistry of Sanger sequencing Dye-labelled dideoxynucleotide triphosphate Copied strandTemplate strand
- 37. Chemistry of Sanger sequencing Dye-labelled dideoxynucleotide triphosphate Copied strandTemplate strand
- 38. Chemistry of Sanger sequencing Copied strandTemplate strand
- 39. Chemistry of Sanger sequencing Copied strandTemplate strand
- 40. Chemistry of Sanger sequencing Copied strandTemplate strand
- 43. 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 6 × 108 times to read genome (would take another 95.1 years at this speed!)* *original animation took 5 seconds
- 44. The human genome project Cost: £2,000,000,000 First draft completed: 2000 ‘Finished’: 2003 http://www.c-spanvideo.org/program/157909-1 Started: 1989 (in the USA)
- 45. The human genome project Cost: £2,000,000,000 First draft completed: 2000 ‘Finished’: 2003 http://www.c-spanvideo.org/program/157909-1 Started: 1989 (in the USA) “Without a doubt, this is the most important, most wondrous map ever produced by humankind” W.J. Clinton, 26/06/2000
- 46. The human genome project Cost: £2,000,000,000 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) “Without a doubt, this is the most important, most wondrous map ever produced by humankind” W.J. Clinton, 26/06/2000
- 47. Solexa sequencing • Cost to sequence a human genome: around £700 (compared to £2,000,000,000) • Time to sequence a human genome: less than a day (compared to 14 years) • Based on a microscope that can detect single molecules https://www.genome.gov/sequencingcosts/ Solexa’s first sequencer launched Illumina buys Solexa
- 48. What’s so impressive about detecting a single molecule? All images © OCR, 2011-2014
- 49. What’s so impressive about detecting a single molecule? All images © OCR, 2011-2014
- 50. What’s so impressive about detecting a single molecule? All images © OCR, 2011-2014
- 51. What’s so impressive about detecting a single molecule? All images © OCR, 2011-2014
- 52. What’s so impressive about detecting a single molecule? All images © OCR, 2011-2014
- 53. What’s so impressive about detecting a single molecule? All images © OCR, 2011-2014
- 54. Looking for a needle in a haystack? What’s so impressive about detecting a single molecule?
- 55. Looking for a needle in a haystack? How many blades of grass on a football pitch? What’s so impressive about detecting a single molecule?
- 56. Looking for a needle in a haystack? About 200,000,000 or 2×108 How many blades of grass on a football pitch? What’s so impressive about detecting a single molecule?
- 57. 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? What’s so impressive about detecting a single molecule?
- 58. 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? What’s so impressive about detecting a single molecule?
- 59. 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 What’s so impressive about detecting a single molecule?
- 60. 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... What’s so impressive about detecting a single molecule?
- 61. 1 mole of grass blades = 3×1015 football pitches = 15×1012 km2 What’s so impressive about detecting a single molecule?
- 62. 1 mole of grass blades = 3×1015 football pitches = 15×1012 km2 Surface area of Earth = 5×108 km2 (1011 football pitches!) What’s so impressive about detecting a single molecule?
- 63. 1 mole of grass blades = 3×1015 football pitches = 15×1012 km2 Surface area of Jupiter = 6×1010 km2 What’s so impressive about detecting a single molecule?
- 64. 1 mole of grass blades = 3×1015 football pitches = 15×1012 km2 Surface area of the Sun = 6×1012 km2 What’s so impressive about detecting a single molecule?
- 65. 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! What’s so impressive about detecting a single molecule?
- 66. 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 What’s so impressive about detecting a single molecule?
- 67. Sanger sequencing Sanger sequencing uses about 2×1010 molecules per 100 letters
- 68. Sanger sequencing Sanger sequencing uses about 2×1010 molecules per 100 letters
- 69. 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: Total Internal Reflection Fluorescence (TIRF) microscopy
- 70. 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: Total Internal Reflection Fluorescence (TIRF) microscopy
- 73. Solexa sequencing © Royal Society of Chemistry publishing
- 74. Solexa sequencing © Royal Society of Chemistry publishing
- 75. Solexa sequencing © Royal Society of Chemistry publishing
- 76. Solexa sequencing © Royal Society of Chemistry publishing Genome Research Ltd.
- 77. Solexa sequencing © Royal Society of Chemistry publishing Genome Research Ltd. Cost to sequence a human genome: around £700 Time to sequence a human genome: less than a day First African, Asian and giant panda genomes sequenced Sanger Institute owns 37 instruments
- 78. Solexa sequencing © Royal Society of Chemistry publishing Genome Research Ltd. Cost to sequence a human genome: around £700 Time to sequence a human genome: less than a day First African, Asian and giant panda genomes sequenced Sanger Institute owns 37 instruments
- 79. Solexa sequencing chemistry deoxynucleotide triphosphate Dye-labelled dideoxynucleotide triphosphate Sanger Sequencing Solexa Sequencing Reversible dye- terminator triphosphate
- 80. Chemistry of Solexa sequencing Copied strandTemplate strand
- 81. Chemistry of Solexa sequencing Copied strandTemplate strand Reversible dye-terminator
- 82. Chemistry of Solexa sequencing Copied strandTemplate strand Reversible dye-terminator
- 83. Chemistry of Solexa sequencing Copied strandTemplate strand
- 84. Chemistry of Solexa sequencing Copied strandTemplate strand Triphenyl phosphine (reacts with azides)
- 85. Chemistry of Solexa sequencing Copied strandTemplate strand
- 86. Summary
- 87. 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 £2 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 the chemistry department in 1997 is now widely used for sequencing worldwide The cost of sequencing has fallen to £700 and takes less than a day
- 88. 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 £2 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 the chemistry department in 1997 is now widely used for sequencing worldwide The cost of sequencing has fallen to £700 and takes less than a day
- 92. Chemists Is it… chemistry? Molecular Biologists/biochemists Physicists/Physical Chemists/Engineers Software engineers/ bioinformaticians