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Sequence information can be obtained from single DNA molecules

Sequence information can be obtained from single DNA molecules. Ido Braslavsky, Benedict Hebert, Emil Kartalov Stephen R. Quake. PNAS, 100(7):3960-3964 (2003). Article critique presented by Véronique Lecault January 30 th , 2007 EECE 491c.

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Sequence information can be obtained from single DNA molecules

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  1. Sequence information can be obtained from single DNA molecules Ido Braslavsky, Benedict Hebert, Emil Kartalov Stephen R. Quake PNAS, 100(7):3960-3964 (2003) Article critique presented by Véronique Lecault January 30th, 2007 EECE 491c

  2. The 1000$ Genome Challenge $3 billion Human genome project: $30 million Current cost for mammalian genome: $100 000 2010 target: $1000 2015 target: “Remember that time is money” -Benjamin Franklin, 1748 Braslavsky et al. (2003) PNAS 100(7):3960-3964

  3. Routes to Genomic Sequence Source of DNA Amplification of a single DNA copy DNA amplification and/or modification Sequencing 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 Braslavsky et al. (2003) PNAS 100(7):3960-3964

  5. The Experimental System Braslavsky et al. (2003) PNAS 100(7):3960-3964

  6. Results Braslavsky et al. (2003) PNAS 100(7):3960-3964

  7. Results • Expose primer • Photobleach primer • Flow dUTP-Cy3 + polymerase and wash out • Expose with green laser • Flow dCTP-Cy5 + polymerase and wash out • Expose with red laser • Flow dATP and dGTP + polymerase and wash out • Flow dCTP + polymerase and wash out • Expose with red laser Braslavsky et al. (2003) PNAS 100(7):3960-3964

  8. Results – Template 1 Yield : 50% Braslavsky et al. (2003) PNAS 100(7):3960-3964

  9. Results – Template 3 Yield : 10% Braslavsky et al. (2003) PNAS 100(7):3960-3964

  10. Results – Templates 1 & 2 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 Braslavsky et al. (2003) PNAS 100(7):3960-3964

  12. Critique Summary • 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 Major points • 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 Minor points Braslavsky et al. (2003) PNAS 100(7):3960-3964

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