single molecule dna manipulations
Download
Skip this Video
Download Presentation
Single-molecule DNA Manipulations

Loading in 2 Seconds...

play fullscreen
1 / 38

Single-molecule DNA Manipulations - PowerPoint PPT Presentation


  • 134 Views
  • Uploaded on

Single-molecule DNA Manipulations. Course Overview. Day 1: Techniques & Basic Results Day 2: Twisting DNA molecules Day 3: DNA and RNA polymerases Day 4: DNA topoisomerases Day 5: DNA packaging. Day 1: Techniques & Basic Results. Historic overview & Introduction

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Single-molecule DNA Manipulations' - brittany


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
course overview
Course Overview
  • Day 1: Techniques & Basic Results
  • Day 2: Twisting DNA molecules
  • Day 3: DNA and RNA polymerases
  • Day 4: DNA topoisomerases
  • Day 5: DNA packaging
day 1 techniques basic results
Day 1: Techniques & Basic Results
  • Historic overview & Introduction
  • Measurement techniques, calibration, noise
  • Stretching nucleic acids
  • Single-molecule DNA sequencing?
  • Zero-force experiments (fluorescence measurements)
length energy and force scales

Bacteria

~eV

~kBT

(4 x 10-21 J)

or 4 pN nm

(1.6 x 10-19 J)

Length, energy- and force-scales

Energies

rotation of the f1 atpase
Rotation of the F1-ATPase

Noji et al., Nature (1997) 386: 299-302.

single molecule paradigm
Object localized in space

Real-time readout

“Synchronized”

Reversible

Object freely diffusing

Offline readout

“Unsynchronized”

Irreversible

Single-molecule paradigm

Single-molecule assay

Bulk biochemical assay

instruments
Fixed-position

Atomic Force Microscope

Micropipette

Optical Tweezer

Fixed-force

Magnetic Trap

Instruments

Feedback loop can convert one into the other

atomic force microscope
Atomic Force Microscope

Cantilevers:

~50-100 mm long, 30 mm wide, 0.2 mm thick

High spatial positionning accuracy (0.1 nm)

Very stiff (5-100 pN/nm) cantilever

High-force instrument (~10 pN-1 nN)

High bandwidth (~kHz in water)

Large size and high bandwidth lead to large noise:

5-10 pN rms noise with 1 kHz bandwidth

High noise is due to size ( ~0.5 pN/Hz-1/2) smaller cantilevers being developped

  • Measurement modes (imaging, stretching)
micropipette
Micropipette

Weaker cantilever (~2 pN/mm)

Larger cantilever, lower bandwidth

 lower force noise than AFM

High forces achievable

Rotation also possible (Bustamante, Heslot)

optical trap
Optical Trap

Relatively stiff (~0.1-1 pN/nm)

High Forces (~100 pN)

Small beads (~0.5 mm)

High bandwidth (~kHz)

Low force noise

Rotation possible but difficult

Bead trapped at beam waist

Higher dielectric than water needed

magnetic tweezer
Magnetic Tweezer

Very weak stiffness of “trap” (pN/mm)

 results in a “constant force” mode

 requires a stiff tether

Force depends on bead size:

1 mm dia bead ~ 1 pN

2.8 mm dia bead ~ 15 pN

4.5 mm dia bead ~ 80 pN

Bandwidth depends on bead size

Low force noise, ultra stable and very low drift

Rotation easy

other techniques
Other techniques
  • Flow fields
    • Force changes along DNA
    • Costly in protein
  • Electric fields
    • Force changes along DNA.
force calibration
Force Calibration
  • Calibration against flow field (F=6phrv)
  • Micropipettes/AFM cantilevers can be calibrated using a set of levers of decreasing stiffness
  • Trap stiffness in all cases easily determined by analyzing Brownian motion
brownian motion analysis

Fx= Fsinq ~ Fq ~ F

dx

__

l

F

F

__

__

l

l

1

1

_

_

<dx2> = kBT

kx<dx2> = kBT

2

2

Brownian Motion Analysis

(Tethered bead in a harmonic potential)

_

Fx = dx = kxdx

Equipartition of energy:

kBT l

____

F =

<dx2>

signal to noise
Signal-to-noise

Thermal agitation causes the mean force to fluctuate with a variance

<dF2> = 4kBT 6phr Df

For a ~1 mm diameter bead in water (h = 10-3 poise) at room temperature,

dF ~ 10 fN/Hz1/2

The detector (bead, cantilever) undergoes rms fluctuations

in its mean position:

dz = dF/kz

(for DNA in our experiments k ~ 10-8 to 10-7 N/m)

dz ~ tens of nm with ~1 s averaging

  • To reduce noise, three approaches (each with its own problems):
  • Average longer
  • Smaller detector
  • Stiffer (i.e. shorter) DNA or polymer
polymer springs

l

3

_

k BT

___

_

2

x

l0

Polymer Springs
  • Polymer elasticity characterized by
    • Persistence length x = A/kBT
    • Relative extension l/l0
  • Simplest case: random walk (Freely-Jointed Chain, FJC)
    • Fully flexible joints between persistence-length units (no bending energy)
    • Entropic elasticity at low force: F=
    • High force is like aligning spin with mag.field
freely jointed chain vs worm like chain
Freely-Jointed Chain vs. Worm-like chain

A: entropic

B: enthalpic

C: “overstretch”

1st measurement: Finzi et al., Science (1992) 258:1122-6.

effect of ionic conditions on x
Effect of ionic conditions on x

Fit is to Poisson-Boltzmann model for uniformly charged cylinder

Baumann et al., PNAS (1997) 94:6185-90.

b s transition and ssdna
BS transition and ssDNA

C

B

D

A

Cluzel et al., Science (1996) 271:792-4.

Cui et al., “ “ “ 795-9.

protein elasticity unfolding titin
Protein elasticity:unfolding titin

Rief et al., Science (1997) 276 :1109-12.

stretching ssdna
Stretching ssDNA

Dessinges et al., PRL (2002) 89, 248102.

base pairing disrupted by salt or chemical modification
Base-pairing disrupted by salt or chemical modification
  • = 0.8 nm
  • Y ~ 200 Mpa
  • Electrostatics+pairing
  • Self-avoiding

ssDNA FJC:

Dessinges et al., PRL (2002) 89, 248102.

single molecule sequencing
Single-molecule sequencing?
  • l-Exonuclease
  • a-hemolysin/nanopores (D. Branton Harvard/Rowland)
  • Optical waveguides (W. Webb, Cornell U)

Human genome: ~3 Gbp…how to sequence in one hour??

digestion of dna by l exonuclease
Digestion of DNA by l-Exonuclease

“Direct” assay

“Conversion” assay

Perkins et al., Science (2003) 301: 1914-8.

Van Oijen et al., Science (2003) 301:1235-8.

constant force digestion rate
Constant-force digestion rate

Perkins et al., Science (2003) 301: 1914-8.

digestion rate is force independent
Digestion rate is force-independent

Perkins et al., Science (2003) 301: 1914-8.

conversion of dsdna to ssdna
Conversion of dsDNA to ssDNA

Van Oijen et al., Science (2003) 301:1235-8.

waveguide sequencing
Waveguide Sequencing

Levene et al., Science (2003) 299: 682-6.

folding unfolding of structured rna
Folding/unfolding of structured RNA

Liphardt et al., Science (2001) 292: 733-7.

folding unfolding of structured rna31
Folding/unfolding of structured RNA

DG ~ F1/2Dx = 14pN x 20 nm = 280 pNnm = 70 kBT

(or, ~170 kJ/mol)

Liphardt et al., Science (2001) 292: 733-7.

effect of an external potential on rates
Effect of an external potential on rates

Energy landscape

(no external force)

Energy landscape

(including external force)

Energy

Dln

Dld

Potential energy

from external force

Reaction coordinate (distance, l) along stretching force

a(F) = a0 exp(FDln/kBT)

b(F) = b0 exp(-FDld/kBT)

unzipping dna
Unzipping DNA

Essevaz-Roulet et al. PNAS (1997) 94 11935-11940

Spatial resolution ~ 50 nm

Needle stiffness ~ 1.7 pN/mm

If Eu ~ 2kBT and Dxu ~ 2x0.3nm, we expect Fu ~ 13 pN

unzipping signal and force flips
Unzipping signal and force flips

Essevaz-Roulet et al. PNAS (1997) 94 11935-11940

tirf based detection of dna hybridization
TIRF-based detection of DNA hybridization

Singh-Zocchi et al., PNAS (2003) 100: 7605-10.

hybridization signal
Hybridization signal

Singh-Zocchi et al., PNAS (2003) 100: 7605-10.

combining manipulation and visualization actin myosin interaction
Combining manipulation and visualization: actin/myosin interaction

Ishijima et al. Cell (1998) 92:161-71.

ad