using nanopores to sequence dna · the role of dna in biology - each human cell has two homologous...

Using nanopores to sequence DNA

Aleksei Aksimentiev

Wednesday, November 27, 13

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phosphate

sugar ring

base

Double stranded DNA(persist. length ~50nm)

Single stranded DNA(persist. length ~1.5nm)

backboneDNA code is written in atomsHighly charged: 2 electron charges per 0.32nm

The sequence has direction:5’-AAGCTGGTTCAG-3’


Double stranded DNA(persist. length ~50nm)

Single stranded DNA(persist. length ~1.5nm)

Highly charged: 2 electron charges per 0.32nm

The sequence has direction:5’-AAGCTGGTTCAG-3’

DNA code is written in atomsbackbone


The role of DNA in biology

- Each human cell has two homologous copies of the genome (from father and mother), each containing ~3,100,000,000 (3.1x109) base pairs. Fits on a CD. - All cell types in a human body have the same genome, but appear different because different part of the genome are “ON” - Genes (protein coding part) occupy only 1.5% of the sequence. ~23,000 genes, twice as many as in a fruit fly. 50% of the genes have unknown function. - More than 50 percent are simple repeat sequences CGTCGTCGTCGT…, the “dark matter” of the genome. Difficult to characterize.- Among individuals, 99.9% of the sequence is similar. > 1,000,000 differences.

Central dogma of molecular biology


The Human Genome Project

Duration: October 1990 - 2003

Discovered ALL 20,000-25,000 human genes

Determined complete sequence of the 3 billion DNA bases5’-ACCGGTGGGTGCATAGCTGTGCTGTAAGTGAAGTG AGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAG GTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTG GACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTA GTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT


GTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT CCTAGCTAGTCAGTGGTGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGA TCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTATTGTGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGWednesday, November 27, 13

GATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT CCTAGCTAGTCAGTGGTGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGA TCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTATTGTGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGAAACGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTG GCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT CCTAGCTAGTCAGTGGTWednesday, November 27, 13

CAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGATCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTATTGTGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGAAACGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTG GCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT CCTAGCTAGTCAGTGGTGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT CCTAGCTAGTCAGTGGTGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGWednesday, November 27, 13

! and ~ 3,000,000 more pages!

(one month to show 24/7)

Just four letter:ACGT

~715 Mb

0 01 1

2 bits

8 bits = 1b

4/8*3*10^9DNA code is billion times moreefficient


2006

2008

2010

$1,000,000

$100,000

Disruptive technologies emerged in: -2006 (Generation II) -2009 (Generation III)

$1,000 genomeexpected in 2013


Conventional DNA sequencingNobel Prize in Chemistry 1980

As the DNA is synthesized, nucleotides are added on to the growing chain by the DNA polymerase.

The reactions start from the same nucleotide and end with a specific base

Fluorescence-based sequence gelhttp://bbrp.llnl.gov


Electric readout of the DNA sequencing

A. Aksimentiev J.P. LeburtonK. SchultenS. SligarG. Timp

(2003)


Coulter counter

patented in 1953 by W.H. Coulter

1/current (i.e. resistance) measurements when a particle enters the microscopic pore.

folk.uio.no/anderne/research.html


The nanopore technology

Distributions of the electric field

A voltage bias is imposed across the membrane

Ionic current

time

I

+ + + + + +

- - - - - - - - -


Ionic current through pore measured

current transients associated with

passage of dsDNA

- Isolates 1nm3 of volume- Automatic loading and reloading- Highly processive, single-file transport- Compatible with several detection schemes- No limit on the read length




3 nm1000 times per minute


The ionic current blockade reveals the sequence of the confined nucleotides

Sequencing DNA by measuring ionic current

Nature Reviews Drug Discovery 1, 77-‐84 (January 2002)


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Nature Biotech. 30: 349 - 353 (2012)

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Sequencing DNA using MspA


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All-Atom Molecular Dynamics Simulations of Nanopore Systems

Massive parallel computerBlue Waters (UIUC): ~200,000 CPUs

Atoms move according to classical mechanics (F= ma)

Interaction between atoms is defined by molecular force field

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Time scale: ~ 0.1-100 µsLength scale: 10K - 100M atoms or (< 50 nm)3

Time resolution: 2 fsSpacial resolution: 0.1 A


Setting up an simulation is like cooking


-‐ protein

-‐ DNA

-‐ lipid

-‐ ions

-‐ water

Components

F = ma @ 300 K

Time step = 1 fs

Setting up an simulation is like cooking


CompuEng conductance of α-‐hemolysin with molecular dynamics

Protein + lipid bilayer membrane + 1M water solution of KCl = ~300,000 atoms

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tric

field

Average electrostatic potential map


Instantaneous current

Current-voltage curve of !-hemolysin

Scope is the current

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Instantaneous current

Ionic current at 120mV: Experiment: 120 pA Simulations: 130 pA

Current-voltage curve of !-hemolysin!"#$%&'()*("<<P`:=a">ABBaC


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Simulated on D. E. Shaw Research computer system Anton

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Electric readout of the DNA sequencing

A. Aksimentiev J.P. LeburtonK. SchultenS. SligarG. Timp

(2003)


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• DNA + nanopore

• water + DNA + nanopore

• ions + water + DNA + nanopore

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Microscopic model of a nanopore

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User-Defined Forces in NAMD

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Medium Medium Advanced

Estimated completion time: ~ 2 hours/sectionTclBC and TclForces: basic knowledge of Tcl

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The best of the two worlds

Solid-state pore Biological pore Hybrid pore?

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Using nanopores to trap DNA

2.5 nmText

Jeff Comer


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Using nanopores to trap DNA

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Graphene nanopores

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b

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Plasmonics tweezers for nanopore sensing

54

Experiment: Cees Dekker, Magnus Jonsson (TU Delo)


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using nanopores to sequence dna · the role of dna in biology - each human cell has two homologous...

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