1. Sequence Information can be Obtained from Single DNA Molecules I. Braslavsky, B. Hebert, E. Kartalov and S. R. Quake (2003) PNAS 100, 3960-64 Eryang Li March 11 2004

2. Why sequence single DNA molecules? Single-molecule studies can provide information about complex biological molecules and systems that is difficult to obtained from ensemble. Single-molecule studies can provide information about complex biological molecules and systems that is difficult to obtained from ensemble. Single-molecule methods can study fluctuating systems under equilibrium conditions. Single-molecule methods can study fluctuating systems under equilibrium conditions. Single-molecule methods can measure time trajectories and reaction pathways of individual members in a nonequilibrated system. Single-molecule methods can measure time trajectories and reaction pathways of individual members in a nonequilibrated system. Single molecules sequencing provides high sensitivity, low cost, fast and maybe sufficient. Single molecules sequencing provides high sensitivity, low cost, fast and maybe sufficient.

3. Sequencing Sanger method of DNA sequencing Sanger method of DNA sequencing cDNA sequencing is used to determine exon splicing patterns and as a tool to discover gene function from context-specific expression data. cDNA sequencing is used to determine exon splicing patterns and as a tool to discover gene function from context-specific expression data. The sequencing technologies based on single molecule measurement is to observe the interaction of particular proteins with DNA or to use ultra high-resolution scanned probe microscopy. The sequencing technologies based on single molecule measurement is to observe the interaction of particular proteins with DNA or to use ultra high-resolution scanned probe microscopy. This work used a combination of evanescent wave microscopy and single-pair fluorescence resonance energy transfer (spFRET) to reject unwanted noise. This work used a combination of evanescent wave microscopy and single-pair fluorescence resonance energy transfer (spFRET) to reject unwanted noise.

4. Fluorescence Resonance Energy Transfer Fluorescence Resonance Energy Transfer (FRET) between two dyes, donor and acceptor, is a powerful technique for studying conformational distribution and dynamics of biological molecules. Fluorescence Resonance Energy Transfer (FRET) between two dyes, donor and acceptor, is a powerful technique for studying conformational distribution and dynamics of biological molecules. FRET, detected at the single-molecule level, opens up new opportunities to probe the detailed kinetics of structural changes without the need for synchronization. FRET, detected at the single-molecule level, opens up new opportunities to probe the detailed kinetics of structural changes without the need for synchronization. Single-pair FRET(spFRET) can report on dynamical changes in the distance or orientation between two fluorophores for intramolecular and intermolecular FRET. Single-pair FRET(spFRET) can report on dynamical changes in the distance or orientation between two fluorophores for intramolecular and intermolecular FRET. Ha T. (2001) Methods 25: 78-86

5. Physical observable Intramolecular detection of conformational changes by spFRET Dynamic colocalization and detection of association by intermolecular spFRET ID, IA – donor and acceptor emission intensities Physical observable Intramolecular and intermolecular spFRET nuclease-DNA interactions Weiss S. (1999) Science 283: 1676-83

6. DNA polymerase DNA polymerase are responsible for the synthesis of new DNA strand on a single-stranded (ss) template. DNA polymerase are responsible for the synthesis of new DNA strand on a single-stranded (ss) template. DNA polymerases play a key role in the replication, repair, and proofreading of DNA by catalyzing the addition of a complementary dNTP to the 3’ end of the growing strand. DNA polymerases play a key role in the replication, repair, and proofreading of DNA by catalyzing the addition of a complementary dNTP to the 3’ end of the growing strand. DNA polymerase enzyme can operate with high fidelity and discrimination when using the modified nucleotide triphosphates and anchored DNA templates. DNA polymerase enzyme can operate with high fidelity and discrimination when using the modified nucleotide triphosphates and anchored DNA templates. Maier B. (2000) PNAS 97: 12002-7

7. The optical setup cy3cy5

8. Sequencing single molecules with FRET dUTP-cy3+polymerase Circle of dUTP-cy3, dCTP-cy3, dATP, dGTP dATP,dGTP+polymerase U-Cy5 dATP,dGTP+polymerase C-Cy5

9. Correlation between the locations of the DNA templates and labeled nucleotides Positions of the fluorescent molecules on the surface were compared with the positions of the DNA molecules A high correction between the primer position and the nucleotide position was found for the correct match. A correlogram in which the positions of detected molecules in the two fields of view are cross-correlated with each other. The locations of DNA templates The labeled nucleotides

10. spFRET monitors the incorporation of nucleotides in the templates The polymerase refused to incorporate C-Cy3 Correctly incorporated U-Cy3 Fill in gap with A and G by FRET The polymerase refused to incorporate U-Cy5 Correctly incorporated C-Cy5

11. Intensity trace from single template molecule and the FRET efficiency

12. Sequence space for 4-mers composed of A and G

13. The incorporation yield of the labeled nucleotides is largely determined by the interaction of the DNA polymerase with modified nucleotide triphosphates. Adjacent incorporation

14. Summary DNA polymerase is active on surface-immobilized DNA templates and can incorporate nucleotides with high fidelity. DNA polymerase is active on surface-immobilized DNA templates and can incorporate nucleotides with high fidelity. With the spFRET method, the sequence information can be obtained single molecule sequence fingerprints up to 5bp in length. With the spFRET method, the sequence information can be obtained single molecule sequence fingerprints up to 5bp in length. The activity of DNA polymerase at the single molecule level provide the foundation for a practical single molecule sequencing technology. The activity of DNA polymerase at the single molecule level provide the foundation for a practical single molecule sequencing technology.

15. Discussion What’s kind of problems are best solved by single- molecule studies? What’s kind of problems are best solved by single- molecule studies? What are the prospects for turning this method into a practical DNA sequencing technology? What are the prospects for turning this method into a practical DNA sequencing technology? Is it a powerful technology for de novo genome sequencing, identity of the expressed gene, and studying basic biochemical questions concerning DNA polymerase activity? Is it a powerful technology for de novo genome sequencing, identity of the expressed gene, and studying basic biochemical questions concerning DNA polymerase activity?