Download de novo

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?

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.
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.
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
Intramolecular and intermolecular spFRET
nuclease-DNA interactions
Physical observable
Intramolecular detection of
conformational changes by
spFRET
Physical observable
Dynamic colocalization and
detection of association by
intermolecular spFRET
Weiss S. (1999) Science 283: 1676-83
ID, IA – donor and acceptor
emission intensities
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
The optical setup
cy3
cy5
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
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.
The labeled nucleotides
• A correlogram in which the
positions of detected
molecules in the two fields of
view are cross-correlated with
each other.
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
Intensity trace from single template molecule
and the FRET efficiency
Sequence space for 4-mers composed
of A and G
Adjacent
incorporation
The incorporation yield of the labeled nucleotides is largely determined by
the interaction of the DNA polymerase with modified nucleotide
triphosphates.
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.
Discussion

What’s kind of problems are best solved by singlemolecule 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?