Sequence Information can Sequence Information can be Obtained from Single be Obtained from Single

DNA MoleculesDNA Molecules

I. Braslavsky, B. Hebert, E. Kartalov and S. R. QuakeI. Braslavsky, B. Hebert, E. Kartalov and S. R. Quake

(2003) (2003) PNASPNAS 100, 3960-64 100, 3960-64

Eryang LiEryang LiMarch 11 2004March 11 2004

Why sequence single DNA molecules?Why sequence single DNA molecules?

Single-molecule studies can provide information about coSingle-molecule studies can provide information about complex biological molecules and systems that is difficult to mplex biological molecules and systems that is difficult to obtained from ensemble. obtained from ensemble.

Single-molecule methods can study fluctuating systems uSingle-molecule methods can study fluctuating systems under equilibrium conditions.nder equilibrium conditions.

Single-molecule methods can measure time trajectories aSingle-molecule methods can measure time trajectories and reaction pathways of individual members in a nonequilnd reaction pathways of individual members in a nonequilibrated system. ibrated system.

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

SequencingSequencing

Sanger method of DNA sequencingSanger method of DNA sequencing

cDNA sequencing is used to determine exon splicing patterns and acDNA sequencing is used to determine exon splicing patterns and as a tool to discover gene function from context-specific expression ds a tool to discover gene function from context-specific expression data. ata.

The sequencing technologies based on single molecule measuremeThe sequencing technologies based on single molecule measurement is to observe the interaction of particular proteins with DNA or to unt is to observe the interaction of particular proteins with DNA or to use ultra high-resolution scanned probe microscopy.se ultra high-resolution scanned probe microscopy.

This work used a combination of evanescent wave microscopy and This work used a combination of evanescent wave microscopy and single-pair fluorescence resonance energy transfersingle-pair fluorescence resonance energy transfer (spFRET) to reje (spFRET) to reject unwanted noise. ct unwanted noise.

Fluorescence Resonance Energy TransferFluorescence Resonance Energy Transfer

Fluorescence Resonance Energy Transfer (FRET) between two dyes, dFluorescence Resonance Energy Transfer (FRET) between two dyes, donor and acceptor, is a powerful technique for studying conformational onor and acceptor, is a powerful technique for studying conformational distribution and dynamics of biological molecules. distribution and dynamics of biological molecules.

FRET, detected at the single-molecule level, opens up new opportunitieFRET, detected at the single-molecule level, opens up new opportunities to probe the detailed kinetics of structural changes without the need fs to probe the detailed kinetics of structural changes without the need for synchronization. or synchronization.

Single-pair FRET(spFRET) can report on dynamical changes in the distSingle-pair FRET(spFRET) can report on dynamical changes in the distance or orientation between two fluorophores for intramolecular and intance or orientation between two fluorophores for intramolecular and intermolecular FRET. ermolecular FRET.

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

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 nuIntramolecular and intermolecular spFRET nuclease-DNA interactionsclease-DNA interactions

Weiss S. (1999) Science 283: 1676-83

DNA polymeraseDNA polymerase DNA polymerase are responsible for the synthesis of new DNA polymerase are responsible for the synthesis of new

DNA strand on a single-stranded (ss) template.DNA strand on a single-stranded (ss) template.

DNA polymerases play a key role in the replication, repair, DNA polymerases play a key role in the replication, repair, and proofreading of DNA by catalyzing the addition of a cand proofreading of DNA by catalyzing the addition of a complementary dNTP to the 3’ end of the growing strand. omplementary dNTP to the 3’ end of the growing strand.

DNA polymerase enzyme can operate with high fidelity anDNA polymerase enzyme can operate with high fidelity and discrimination when using the modified nucleotide triphod discrimination when using the modified nucleotide triphosphates and anchored DNA templates. sphates and anchored DNA templates.

Maier B. (2000) PNAS 97: 12002-7

The optical setupThe optical setup

cy3 cy5

Sequencing single molecules with FRETSequencing single molecules with FRET

dUTP-cy3+polymerase

Circle of dUTP-cy3, dCTP-cy3, dATP, dGTP

dATP,dGTP+polymeraseU-Cy5

dATP,dGTP+polymeraseC-Cy5

Correlation between the locations of the DNA Correlation between the locations of the DNA templates and labeled nucleotidestemplates 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

spFRET monitors the incorporation of nucleospFRET monitors the incorporation of nucleotides in the templatestides 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

Intensity trace from single template molecule Intensity trace from single template molecule and the FRET efficiencyand the FRET efficiency

Sequence space for 4-mers composed Sequence space for 4-mers composed of A and Gof A and G

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

Adjacent incorporation

SummarySummary DNA polymerase is active on surface-immobilized DNA tDNA polymerase is active on surface-immobilized DNA t

emplates and can incorporate nucleotides with high fideliemplates and can incorporate nucleotides with high fidelity. ty.

With the spFRET method, the sequence information can With the spFRET method, the sequence information can be obtained single molecule sequence fingerprints up to be obtained single molecule sequence fingerprints up to 5bp in length. 5bp in length.

The activity of DNA polymerase at the single molecule leThe activity of DNA polymerase at the single molecule level provide the foundation for a practical single molecule vel provide the foundation for a practical single molecule sequencing technology. sequencing technology.

DiscussionDiscussion

What’s kind of problems are best solved by single-molecWhat’s kind of problems are best solved by single-molecule studies?ule studies?

What are the prospects for turning this method into a praWhat are the prospects for turning this method into a practical DNA sequencing technology?ctical DNA sequencing technology?

Is it a powerful technology for Is it a powerful technology for de novode novo genome sequenci genome sequencing, identity of the expressed gene, and studying basic bing, identity of the expressed gene, and studying basic biochemical questions concerning DNA polymerase activitochemical questions concerning DNA polymerase activity? y?