dna sequencing methods past, present, and...
TRANSCRIPT
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2255-1
2nd Conference on Systems Biology and New Sequencing Techniques" (2-4 November), preceded by Introductory Lectures on
"Quantitative Approaches to Biological Problems" (31 October - 1 November) November)'
Arjang Hassibi
31 October - 4 November, 2011
University of Texas at Austin USA
DNA Sequencing Methods Past, Present, and Future
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2nd Conference on Systems Biology and New Sequencing Techniques
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A TG CA TA TC GC GC GT AG CT AT A
5’3’
Str
and
(X
)
Str
and
(X
*)
Strand (X) and (X*) arecomplementary
Complementarybase pairs (bps)
3’5’
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… ACTT?GGTCG…
?
?
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… ACTT?GGTCG…
?
?
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ACGTCTTGTGAACAAC
°
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1958 1980
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Sanger, F.; Nicklen, S.; Coulson, A.R., "DNA sequencing with chain-terminating inhibitors“ PNAS, (1977)
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Electro-phoresis Gel
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DNA
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? ? ? ? ? ? ? ? ? ? ? ? ?
? = A ? A
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ACGT
TGCA
ACGTTTTCAATGC…5’3’
5’ 3’
Primer
START
ADD (A) ADD (C) ADD (G) ADD (T)
ADD (A)ADD (C)ADD (G)ADD (T)
Check Reaction Check Reaction Check Reaction Check Reaction
Check Reaction Check Reaction Check Reaction Check Reaction
Unknown Sequence
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? ? ? ? ? ? ? ? ? ? ? ? ?
START
ADD (A) ADD (C) ADD (G) ADD (T)
ADD (A)ADD (C)ADD (G)ADD (T)
No Reaction Check Reaction Check Reaction Check Reaction
Check Reaction Check Reaction Check Reaction Check Reaction
A
A A
A
1
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START
ADD (A) ADD (C) ADD (G) ADD (T)
ADD (A)ADD (C)ADD (G)ADD (T)
No Reaction No Reaction Check Reaction Check Reaction
Check Reaction Check Reaction Check Reaction Check Reaction
C
C C
C
2
? ? ? ? ? ? ? ? ? ? ? ? ?
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START
ADD (A) ADD (C) ADD (G) ADD (T)
ADD (A)ADD (C)ADD (G)ADD (T)
No Reaction No Reaction No Reaction Reaction
Check Reaction Check Reaction Check Reaction Check Reaction
T
T T
T
4
T
A ? ? ? ? ? ? ? ? ? ? ? ?
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START
ADD (A)ADD (C)ADD (G)ADD (T)
Check Reaction Reaction No Reaction No Reaction
G
G G
G
7
TG
ADD (A) ADD (C) ADD (G) ADD (T)
No Reaction No Reaction No Reaction Reaction
A C ? ? ? ? ? ? ? ? ? ? ?
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ACGTTTTCAATGCGGG
A C G T A T C G A T C G A C G T A C G T A C G T A C G T A C G TA……...G..C.4T……...C…………2AT.. G..C……..3G
ACGTTTTCAATGCGGG
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1 M. Ronaghi, M. Uhlén, and P. Nyrén, "A sequencing method based on real-time pyrophosphate,". Science, 1998.
ACGT
TGCA
5’3’
5’ 3’
Primer
Prophosphate (PPi)
1
APS ATP
ATP-Sulfurylase 2
Lucifern ATP
Luciferase 3
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1 M. Margulies + 54 additional coauthors "Genome sequencing in open microfabricated high density picoliter reactors," Nature (2005).
>1 M micro-wells
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1 M. Margulies + 54 additional coauthors "Genome sequencing in open microfabricated high density picoliter reactors," Nature (2005).
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2 Adessi et al., “Solid phase DNA amplification: characterization of primer attachment and amplification mechanisms,” Nucleic Acids Res., (2000).
1 Turcatti et al., “A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis,” Nucleic Acids Res., (2008).
ACGT
TGCA
3’
5’ 3’
PrimerLabelednucleotide added1
A C G T
A
T
Color specifiessequence2
Label is cleaved3
4 Repeat
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1 J. Eid et al., “Real-time DNA sequencing from single polymerase molecules,” Science, 2009.
Inte
nsi
ty
Time
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1 J. Eid et al., “Real-time DNA sequencing from single polymerase molecules,” Science, 2009.
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1 R. Drmanac et al., “Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays,” Science, 2010.
1
2
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Cross-Section
Top View
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1 Clarke et al., “Continuous base identification for single-molecule nanopore DNA sequencing,” Nature Nano, 2009.
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1 Polonsky et al., “Nanopore in metal-dielectric sandwich for DNA position control,” Applied Physics Letters, 2007.
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