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 30 th, 2007 EECE 491c PNAS, 100(7): (2003)

EECE 491c Braslavsky et al. (2003) PNAS 100(7): The 1000$ Genome Challenge Human genome project: Current cost for mammalian genome: 2010 target: 2015 target: $3 billion $30 million $ $1000 “Remember that time is money” -Benjamin Franklin, 1748

EECE 491c Braslavsky et al. (2003) PNAS 100(7): Routes to Genomic Sequence Source of DNA Amplification of a single DNA copy DNA amplification and/or modification Sequencing

EECE 491c Braslavsky et al. (2003) PNAS 100(7): 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): The Experimental System

EECE 491c Braslavsky et al. (2003) PNAS 100(7): Results

EECE 491c Braslavsky et al. (2003) PNAS 100(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): Results – Template 1 Yield : 50%

EECE 491c Braslavsky et al. (2003) PNAS 100(7): Results – Template 3 Yield : 10%

EECE 491c Braslavsky et al. (2003) PNAS 100(7): Results – Templates 1 & 2

EECE 491c Braslavsky et al. (2003) PNAS 100(7): 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): Critique Summary To be improvedGood Major points Minor points 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