molecular biology and history of dna sequencing · history of dna 1957 1961 1970 1971 1977 1983...
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Molecular Biology and History of DNA Sequencing
02-223 Sept. 9 2014
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
http://www.nature.com/scitable/content/dna-is-a-double-helix-24263
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
DNA Polymerase
h"p://www.virology.ws/2009/05/10/the-‐error-‐prone-‐ways-‐of-‐rna-‐synthesis/
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!
Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002.
PCR
• Polymerase Chain ReacJon • Invented in 1983 • DNA polymerase from Thermus aqua+cus • 2.2x105 error rate
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’
Let’s perform paper PCR
Polymerase Chain Reaction (PCR) overview
http://www.accessexcellence.org/RC/VL/GG/polymerase.php
Starting DNA
Final DNA
Polymerase Chain Reaction (PCR) overview
http://www.accessexcellence.org/RC/VL/GG/polymerase.php
http://www.lifetechnologies.com/us/en/home/life-science/pcr/elevate-pcr-research/pcr-video-library/pcr-animation.html
PCR over time
h"p://mtbakerbio.com/sites/default/files/images/RTPCR%20graphSml.gif
Sanger Sequencing
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
Paper sequencing
Now that we have all these strands of DNA whose final base we know, what do we do with them?
Gel Electrophoresis
ATGGACCAGTTG ATGGACCAGTT ATGGACCAGT ATGGACCAG ATGGACCA ATGGACC ATGGAC ATGGA ATGG ATG AT A
A=green G=yellow
T=red C=blue
HGP and Celera
ABI 3730x (Sanger)
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
Questions?