DNA Sequencing using Nano-sized Pores

Ricardo Urquidi
Bionanotechnology 2011

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Introduction

 Why Nanopore Sequencing?

 Promise of Rapid DNA sequencing
 Nanopores in the 3-1.5 nm range ideally
 Either Natural or synthetic
 ~120mV currents drive DNA movement

Fologea et al. 2005

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Natural Pores

 Most widely used is α-hemolysin in a lipid bilayer

 Only accepts ssDNA pore size under 2nm
 Very stable protein, major draw back is the membrane lifetime

Unblocked Blocked
Branton et al. 2008
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Solid State Pores

 Created from different silicon products (SiO2, SiN4, doped silicon and
metals)
 Can be made through several processes:
 Ion beam sculpting
 e-beam drilling
 Atomic layer deposition

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Measurement Method

 In the traditional approach to nucleotide sequencing a current drop is

measured when the DNA passes through the pore.

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Problems with the Traditional Method

 Multiple problems
 Poor definition between different base pairs
 Rate of DNA translocation through the pore is too high. 1,000,000-50,000 bp/s
The change in current is only in pA range. Need MHz speed for pA measurements.
 Life time of natural membranes
 Fabrication of solid state pores (in lab research)
 Signal to noise ratio

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Slowing DNA translation rate

 Viscosity, PH, Temperature, and Voltage

 Addition of cyclodextrin integrated into natural pores
 Polymerases, and exogenases
 Single dNTP detection
 Pore functionalization

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TEM Nanopores

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TEM Nanopores

 Advantage: nA signal is easier to detect

 Disadvantage: Needs ~1 ms per nucleotide to measure
 Different orientations lead to different measurements

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Functionalized Nanopores

 Orient and help hold DNA in a particular orientation.

 Functionalized carbon nanotubule inserted into pore
 Functionalized electrodes for TEM (Thiolated tips):
One tip for the phosphate “backbone” and an individual tip for each base.

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More Recent Approches

 Capacitance instead of current as the measurable quantitiy

 Hybridization of DNA
 Two technique method:
 Fluorescent labeling and pore

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Take Home Message

 Although nanopore sequencing holds great promise in concept, the

application has been hard.
 Advances are constant but slow with the main challenge being
technological development.
 Several novel ideas are presented to augment the traditional approach,
but all still have several drawbacks, although TEM nonopores are
promising
 Ultimate goal is to produce a rapid sequencing device

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References

 Fologea et al. “Detecting Single Stranded DNA with a Solid State Nanopore” Nano letters 2005 Vol. 5 No. 10

1905-1909

 Michael Zwolak Johan Lagerqvist, Massimiliano Di Ventra “DN, eh? Sequencing via Transverse Electronic

Transport” Powerpoint presentation

 Branton et al. “The potential and challenges of nanopore sequencing” Nature biotechnology 2008, Vol 26 No.

10

 Li et al. “Ion-beam sculpting at nanometre length scales” Nature Vol. 412 12 July 2001

 Fologea et al. “Slowing DNA Translocation in a Solid State Nanopore” Nano Lett. 2005 September ; 5(9)

 Chen et al. “Atomic Layer Deposition to Fine-Tune the Surface Properties and Diameters of Fabricated

Nanopores” Nano letters 2004 Vol 4 No. 7 1333-1337

 Ivanov et al. “DNA Tunneling Detector Embedded in a Nanopore” Nano letters 2011 Vol. 11 279-285

 Chu et al.“Real-Time Monitoring of DNA Polymerase Function and Stepwise Single-Nucleotide DNA Strand

Translocation through a Protein Nanopore” Angew. Chem. 2010 Vol. 49 10106-10109

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