1. Detecting single molecules and
sequencing DNA
Rohan T. Ranasinghe
University Chemical Laboratories, Lensfield Road, Cambridge CB2 1EW

2. Locations and timeline
1 mile

3. Locations and timeline
1 mile
http://www.cambridge
2000.com
Old Cavendish
Laboratory
1953: Discovery of the
structure of DNA

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. 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. 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. 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. 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. 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. 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. 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. DNA Sequencing
Why would you want to sequence DNA?
http://www.sikeston.k12.mo.us

13. DNA Sequencing
Why would you want to sequence DNA?
http://www.sikeston.k12.mo.us
© Invitrogen

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. 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. 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. 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. 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. 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. Sanger sequencing
CAGTCAGTCA
GA
C
G
A
C
TA
G
T
C
Based on copying of DNA:
Genome Research Ltd.

21. Sanger sequencing
CAGTCAGTCA
GA
C
G
T
A
C
TA
G
T
C
Based on copying of DNA:
Genome Research Ltd.

22. Sanger sequencing
CAGTCAGTCA
GA
C
G
T
A
C
TA
CG
T
C
Based on copying of DNA:
Genome Research Ltd.

23. Sanger sequencing
CAGTCAGTCA
GA
C
G
T
A
C
TA
CG
T
A
C
Based on copying of DNA:
Genome Research Ltd.

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. 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. 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. 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. 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. The human genome project
http://www.c-spanvideo.org/program/157909-1
Started: 1989 (in the USA)

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. The human genome project
First draft completed: 2000
‘Finished’: 2003
http://www.c-spanvideo.org/program/157909-1
Started: 1989 (in the USA)

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. 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. 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. 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. What does it mean to detect a single molecule?
Looking for a needle in a haystack?

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. 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. 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. 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. 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. 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. What does it mean to detect a single molecule?
1 mole of grass blades = 3×1015 football pitches = 15×1012 km2

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. 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. 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. 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. 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. Sanger sequencing
Sanger sequencing uses about 2×1010 molecules per 100 letters

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. 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. Solexa sequencing

53. Solexa sequencing
© Royal Society of Chemistry publishing
Densely packed microscopic “islands” of
DNA generate information very quickly

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. Solexa sequencing
© Royal Society of Chemistry publishing Genome Research Ltd.

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. 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. 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