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Sanger Sequencing
- 1. Molecular Biology and History of DNA Sequencing 02-223 Sept. 9 2014
- 2. History of DNA 1866 1869 1911 1950 1953 Gregor Mendel first described patterns of inheritance Fredrich Miescher first isolated DNA Thomas Morgan first described linkage and recombination Edwin Chargaff discovered that A and T, and G and C have equal amounts James Watson and Francis Crick proposed that DNA is a double strand with a double helical structure
- 4. History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in- vitro using DNA polymerase Hamilton Smith discovered DNA restriction enzymes First genome sequenced using in-vitro replication by Ray Wu, A.D. Kaiser, and Ellen Taylor . Phage λ, ~5000 nt took over 3 years Frederick Sanger developed dideoxy DNA sequencing ~100 bases/reaction PCR developed by Kary Mullis Leroy Hood developed automated sequencing Commercial automated DNA synthesizer ~1000 bases/ reaction
- 7. h"p://www.virology.ws/2009/05/10/the-‐error-‐prone-‐ways-‐of-‐rna-‐synthesis/ Even with proofreading, mistakes made every 107-‐109 bases 6 billion bases in human genome!
- 8. Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002.
- 9. PCR • Polymerase Chain ReacJon • Invented in 1983 • DNA polymerase from Thermus aqua+cus • 2.2x105 error rate
- 10. Polymerase Chain Reaction (PCR) overview DNA sample 5’ 3’ 3’ 5’ Separate DNA strands 5’ 3’ 3’ 5’ Melt: ~94oC 30 sec Extend from primers 5’ 3’ 3’ 5’ Extend: 72oC 30 sec/kb + buffer, ssDNA primers, dNTPs, DNA polymerase (Taq) Mg2+ - enzyme cofactor Melt Anneal Extend 25-35 cycles x 5’ 3’ 3’ Hybridize ssDNA primers Anneal: Tm - 5oC 30 sec 5’
- 11. Let’s perform paper PCR
- 13. Polymerase Chain Reaction (PCR) overview http://www.accessexcellence.org/RC/VL/GG/ polymerase.php Starting DNA Final DNA
- 14. Polymerase Chain Reaction (PCR) overview http://www.accessexcellence.org/RC/VL/GG/ polymerase.php
- 19. Following growing DNA strand with ddNTPs ddATP All 4 dNTPs added to each. 10% of the following ddNTP added as well ddGTP ddTTP ddCTP At any base that complements the ddNTP, 10% chance of terminating
- 20. Paper sequencing Now that we have all these strands of DNA whose final base we know, what do we do with them?
- 29. HGP and Celera ABI 3730x (Sanger)
- 30. Pros and Cons of SS • Polymerase errors average out • Long sequences (~450 bp) • Can only do 1 sequence at a time • Need a lot of DNA to start with • Expensive: 2¢/base
- 31. Questions?