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

2nd Conference on Systems Biology and New Sequencing Techniques

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’

… ACTT?GGTCG…

?

?

… ACTT?GGTCG…

?

?

ACGTCTTGTGAACAAC

°

1958 1980

Sanger, F.; Nicklen, S.; Coulson, A.R., "DNA sequencing with chain-terminating inhibitors“ PNAS, (1977)

Electro-phoresis Gel

DNA

? ? ? ? ? ? ? ? ? ? ? ? ?

? = A ? A

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

? ? ? ? ? ? ? ? ? ? ? ? ?

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

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

? ? ? ? ? ? ? ? ? ? ? ? ?

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 ? ? ? ? ? ? ? ? ? ? ? ?

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 ? ? ? ? ? ? ? ? ? ? ?

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

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

1 M. Margulies + 54 additional coauthors "Genome sequencing in open microfabricated high density picoliter reactors," Nature (2005).

>1 M micro-wells

1 M. Margulies + 54 additional coauthors "Genome sequencing in open microfabricated high density picoliter reactors," Nature (2005).

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

1 J. Eid et al., “Real-time DNA sequencing from single polymerase molecules,” Science, 2009.

Inte

nsi

ty

Time

1 J. Eid et al., “Real-time DNA sequencing from single polymerase molecules,” Science, 2009.

1 R. Drmanac et al., “Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays,” Science, 2010.

1

2

Cross-Section

Top View

1 Clarke et al., “Continuous base identification for single-molecule nanopore DNA sequencing,” Nature Nano, 2009.

1 Polonsky et al., “Nanopore in metal-dielectric sandwich for DNA position control,” Applied Physics Letters, 2007.

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