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Sequence Information can be Obtained from Single DNA Molecules

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. Why sequence single DNA molecules?.

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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 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 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 molecules sequencing provides high sensitivity, low cost, fast and maybe sufficient.

  3. 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. • 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.

  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. • 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. Ha T. (2001) Methods 25: 78-86

  5. Physical observable Physical observable Intramolecular and intermolecular spFRET nuclease-DNA interactions Intramolecular detection of conformational changes by spFRET Dynamic colocalization and detection of association by intermolecular spFRET ID, IA – donor and acceptor emission intensities 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 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. Maier B. (2000) PNAS 97: 12002-7

  7. The optical setup cy3 cy5

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

  9. Correlation between the locations of the DNA templates and labeled nucleotides The locations of DNA templates • 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 labeled nucleotides

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

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

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

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

  14. Summary • 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. • 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 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?

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