Sequence information can be obtained from single DNA molecules

Ido Braslavsky, Benedict Hebert, Emil Kartalov Stephen R. Quake

Article critique presented by Véronique Lecault

January 30th, 2007 EECE 491c

PNAS, 100(7):3960-3964 (2003)

EECE 491c Braslavsky et al. (2003) PNAS 100(7):3960-3964 2

The 1000$ Genome Challenge

Human genome project:

Current cost for mammalian genome:

2010 target:

2015 target:

$3 billion

$30 million

$100 000

$1000

“Remember that time is money”

-Benjamin Franklin, 1748

EECE 491c Braslavsky et al. (2003) PNAS 100(7):3960-3964 3

Routes to Genomic SequenceSource of DNA

Amplification of a single DNA copy

DNA amplification and/or modification

Sequencing

EECE 491c Braslavsky et al. (2003) PNAS 100(7):3960-3964 4

What are the advantages of single molecule sequencing?

Individual fragments of DNA do not need to be amplified.

Sequencing can be in real-time (reading do not become dephased)

Low volumes required

Massive parallelization possible

EECE 491c Braslavsky et al. (2003) PNAS 100(7):3960-3964 5

The Experimental System

EECE 491c Braslavsky et al. (2003) PNAS 100(7):3960-3964 6

Results

EECE 491c Braslavsky et al. (2003) PNAS 100(7):3960-3964 7

Results

1. Expose primer

2. Photobleach primer

3. Flow dUTP-Cy3 + polymerase and wash out

4. Expose with green laser

5. Flow dCTP-Cy5 + polymerase and wash out

6. Expose with red laser

7. Flow dATP and dGTP + polymerase and wash out

8. Flow dCTP + polymerase and wash out

9. Expose with red laser

EECE 491c Braslavsky et al. (2003) PNAS 100(7):3960-3964 8

Results – Template 1

Yield : 50%

EECE 491c Braslavsky et al. (2003) PNAS 100(7):3960-3964 9

Results – Template 3

Yield : 10%

EECE 491c Braslavsky et al. (2003) PNAS 100(7):3960-3964 10

Results – Templates 1 & 2

EECE 491c Braslavsky et al. (2003) PNAS 100(7):3960-3964 11

Is That Enough for the Challenge?

Limitations :

FRET readout length (5 nm – 15 pb)

DNA polymerase interaction with modified nucleotides

Far from de novo sequencing with 5 pb and only 2 nucleotides

Hard to assess consecutives bases

EECE 491c Braslavsky et al. (2003) PNAS 100(7):3960-3964 12

Critique Summary

To be improved Good

Ma

jor

po

ints

Min

or

po

ints

•A signature of 5 bp with 2 nucleotides is not very impressive

•More work should be done to improve the yield and allow adjacent incorporations

•Current limitations of this technology are underestimated

• The time and cost of each experiment should be addressed

• Clever way of reducing background fluorescence

• Important step forward in single molecule DNA sequencing

•Solutions suggested to resolve some of the limitations

•Clear representation of FRET signaling•It is not very clear to see what the correlogram correlates

•The paper is disappointing compared to the exciting abstract

•A picture without incorporation is missing for the reader