molecular biology and history of dna sequencing · history of dna 1957 1961 1970 1971 1977 1983...
TRANSCRIPT
![Page 1: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/1.jpg)
Molecular Biology and History of DNA Sequencing
02-223 Sept. 9 2014
![Page 2: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/2.jpg)
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
![Page 3: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/3.jpg)
http://www.nature.com/scitable/content/dna-is-a-double-helix-24263
![Page 4: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/4.jpg)
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
![Page 5: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/5.jpg)
DNA Polymerase
![Page 6: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/6.jpg)
h"p://www.virology.ws/2009/05/10/the-‐error-‐prone-‐ways-‐of-‐rna-‐synthesis/
![Page 7: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/7.jpg)
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!
![Page 8: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/8.jpg)
Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002.
![Page 9: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/9.jpg)
PCR
• Polymerase Chain ReacJon • Invented in 1983 • DNA polymerase from Thermus aqua+cus • 2.2x105 error rate
![Page 10: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/10.jpg)
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’
![Page 11: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/11.jpg)
Let’s perform paper PCR
![Page 12: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/12.jpg)
![Page 13: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/13.jpg)
Polymerase Chain Reaction (PCR) overview
http://www.accessexcellence.org/RC/VL/GG/polymerase.php
Starting DNA
Final DNA
![Page 14: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/14.jpg)
Polymerase Chain Reaction (PCR) overview
http://www.accessexcellence.org/RC/VL/GG/polymerase.php
![Page 15: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/15.jpg)
http://www.lifetechnologies.com/us/en/home/life-science/pcr/elevate-pcr-research/pcr-video-library/pcr-animation.html
![Page 16: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/16.jpg)
PCR over time
h"p://mtbakerbio.com/sites/default/files/images/RTPCR%20graphSml.gif
![Page 17: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/17.jpg)
Sanger Sequencing
![Page 18: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/18.jpg)
![Page 19: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/19.jpg)
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
![Page 20: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/20.jpg)
Paper sequencing
Now that we have all these strands of DNA whose final base we know, what do we do with them?
![Page 21: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/21.jpg)
Gel Electrophoresis
![Page 22: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/22.jpg)
ATGGACCAGTTG ATGGACCAGTT ATGGACCAGT ATGGACCAG ATGGACCA ATGGACC ATGGAC ATGGA ATGG ATG AT A
![Page 23: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/23.jpg)
![Page 24: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/24.jpg)
A=green G=yellow
T=red C=blue
![Page 25: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/25.jpg)
![Page 26: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/26.jpg)
![Page 27: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/27.jpg)
![Page 28: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/28.jpg)
![Page 29: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/29.jpg)
HGP and Celera
ABI 3730x (Sanger)
![Page 30: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/30.jpg)
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
![Page 31: Molecular Biology and History of DNA Sequencing · History of DNA 1957 1961 1970 1971 1977 1983 1986 1996 Marshall Nirenberg elucidated the codons Arthur Kornberg replicated DNA in-vitro](https://reader030.vdocuments.site/reader030/viewer/2022041106/5f07c2577e708231d41e9993/html5/thumbnails/31.jpg)
Questions?