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