molecular biology and history of dna sequencing · history of dna 1957 1961 1970 1971 1977 1983...

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?