Microfluidics for DNA Analysis

8/10/2019 Microfluidics for DNA Analysis

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Microfluidics for DN nalysis

Dr. Thara Srinivasan

Lecture 19

Picture credit: Nanogen

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Lecture Outline

Reading from reader Mastrangelo, C. H. Microfabricated Devices for Genetic

Diagnostics, (1998) pp. 1769-87. Khandurina, J. et al., Bioanalysis in Microfluidic Devices,

(2002) pp. 159-83. Zhang, L., et al., Microchip Electrophoresis-Based

Separation of DNA, (2003) pp. 1645-54.

Todays Lecture DNA and Analysis Methods Scaling in Microfluidics Survey of Microfabricated Chips


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DNA S

B

P 5 3

Genetic information is stored in chromosomes aslong strings of DNA grouped as genes

In humans, 46 chromosomes are 50 - 400 10 6DNA units long (compared to 4 10 6 for E. coli )

Units of DNA are nucleotides, consisting of: A base, a sugar and a phosphate bridge Sugar linkage has directionality, 5 and 3 en ds Four bases: adenine, thymine, guanine, and

cytosine Bases hydrophobic, backbone hydrophilic Single-stranded DNA attaches to complementary

strand (G-C, A-T)

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DNA Analysis DNA is extracted from cell nucleus and purified

Break cell membranes using detergent Remove cell debris, proteins, enzymes

DNA assays Detect specific fragments in fingerprint pattern-matching mode Sequence DNA fragment for base pair order of fragment

Analysis tools Chemical amplification Restriction digestion Electrophoretic separation Sanger sequencing process Hybridization Fluorescence visualization


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Amplification

Animations at: http://www.dnalc.org/resources/BiologyAnimationLibrary.htmhttp://bldg6.arsusda.gov/pberkum/Public/sarl/cregan/pcr.gif

Polymerase chain reaction

Double-stranded DNAdenatured, 95C Primers attach (anneal) to

strands, flanking section tobe amplified, 50-65C

Taq enzymes attach toprimer sites and synthesizenew strands from bases insolution, 72C

Repeat cycle 20-30 timesto get effectiveamplification

Macroscopic thermalcyclers need 90 min peramplification

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Excitation maximumEmission maximum

Detection

Fluorescent labeling with moleculeswhich emit light when excited allowsextremely sensitive visualization offragment

Intercalating dye: ethidium bromide Single fluorophore: fluorescein

Excitation With UV laser-induced fluorescence,

emission signal must be separatedfrom excitation; requires confocalmicroscope

Electrochemiluminescence (ECL)uses Ru(bpy) 2+3 end label, can bedetected with conventional CCD


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Cutting

Restriction digestion isfragmentation of DNA Use restriction nuclease enzymes

to cleave DNA at specific locations(can recognize specific sequencesof 4-8 bases)

Size distribution of restrictionfragments can fingerprint DNAmolecule

Molecular Biology of the Cell

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and Pasting Hybridization is hydrogen bonding

of two complementary singlestrands of DNA Occurs at specific T and salinity

conditions In analyses, known strand is probe,

other is unknown and bindingindicates match

Recognition not perfect, single basemismatches occur

DNA probes immobilized on surfaceusing linker make pixels formicroarrays

Microarray pattern matching

ACGTA

CCGTA GCGTA

TCGTA AGCAT


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Separation Electrophoresis to separate DNA fragments based on size

Mobility EP depends on fragment size and charge and mobile phase DNA fragments in solution are (-) charged and have constant charge

to length ratio Additional molecular sieving matrixes are needed to separate DNA

based on length. Fragments drift in race track where separation is L = EP Et Separation resolution important

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Macroscale Separations

Macroscale gels Thin multilane slabs; preparation is labor-

intensive V up to 2 kV over 20-100 cm Joule heating limits E to 5-40 V/cm Good separation may require hours

Capillary electrophoresis Capillaries 10-300 m in diameter, 50 cm

long Increased surface to volume ratio and

faster heat dissipation permits higher fielduse (up to 1.2 kV/cm)

Good separation in < 1 hour Use of confocal laser-induced

fluorescence Agilent Technologies


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Sequencing Sanger method

Combine PCR andelectrophoretic separations Duplication of DNA

fragment starts at primerlocation, as in PCR

But in addition tonucleotides in solution, alsoadd small amount of dideoxy nucleotides(ddNTPs) of one type (ddA,ddC, ddG, or ddT).

When ddNTP is captured,growing strand terminates,resulting in

complementary strandfragments terminated at allpossible positions for eachbase

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Sequencing

Four-color sequencing Carry out four separate reactions, one for each base. Electrophoretically separate each sample Superimpose results to read out fragment sequence


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Todays Lecture

DNA and Analysis Methods Scaling in Microfluidics Survey of Microfabricated Chips

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Miniaturization Benefits

Benefits Reagent consumption ~ [s 3]

Miniscule reaction volumes reduce reagent cost. Heat transfer ~ [s 2]

Surface phenomena Mass transfer ~ [s 2]

Reduced analysis times, with minimum assay time limited byspeed of enzyme (30-100 bp/s)

Flow is laminar Electroosmotic flow for valveless systems ~ [s 2] Capillary flow ~ [s 1]

Separation efficiency ~ [s -2] Injection volume well-defined


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Miniaturization Issues

Issues Detection limit ~ [s 3],

S/N degraded as [s 3] unless detector area scales withsample [s 1]

Pressure flows ~ [h 3] Other surface phenomena ~ [s 2], [s1]

Wall adsorption effects and sample evaporation [s 2], capillaryforces [s 1]

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Microfluidics Fabrication

Fabrication Batch fabrication

Microchip cost ~ [s 2], but limited by package cost Parallelization to arrays easy Portability increased

Less need for external pumps, detection equipment


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Scaling and Microfluidics

Mastrangelo

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Scaling and Mixing by Diffusion

Mixing by diffusion For channels 1 mm wide and flow velocities 1 cm/s, Re

is low and flow is laminar Time required to travel distance x by diffusion is x 2 /2D For channel width of 70 m and velocity 1 cm/s,

fluorescein ( D = 3 10 -6 cm 2/s) will take 2 s to mix overchannel length of 2 mm

Upper limit of 100 m width for channels


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Scaling and Diffusion Effects

While being carried by electroosmosis and drifted byelectrophoresis, a sample slab can spread out in width due todiffusion

D x w

S S

transit S

transit

L E

DL

U

DL W

D width to grows slab mal infinitesi U L time in

==

=

0

min

0,

If initial slab is smaller than W min , separation is limited only bydiffusion

Ls

U 0

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Scaling and Separation Efficiency

Separation efficiency Number of theoretical plates, N, per unit time

[ ] ][ 1,,

,2

,2

2,

2

2

2

2

2

===

====

s d t

N Ld t V

L U L t

d L

V D V

N E

DL Dt

L N

EK i

EK

x EK

x

x

Resolution parameter, R Peak capacity, n Signal to noise ratio, SNR

l L n

l R x

=

~


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Todays Lecture

DNA analysis methods Scaling in microfluidics Survey of microfabricated devices

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Chip Electrophoresis Capillary electrophoresis (CE) on-

chip First demonstrations in 90s, Manz

group (Imperial College, London) andHarrison group (Univ. of Alberta)

Separation 100 faster than slabgels, 10 faster than CE

CE chips Material ~ glass or plastic Electrodes ~ metal pins inserted into

wells or patterned conductive layer Separation medium ~ chips filled with

unpolymerized liquids are reusable Layout ~ offset double-T Detection ~ confocal fluorescence

microscope focused at single spot

Caliper Technologies


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Parallelization of CEusing arrays for highthroughput

384-channels radialmicroplate forgenotypinganalyses in 98% success

96-channelwafer,Mathies group

UCB

Array CE

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For 10-1000 improved sensitivity, increase sample concentration by Sample stacking Solid-phase extraction

Developments

Santiago group, Stanford

de Rooij group, University of Neuchtel

Injection schemes to givethinner plug

Higher resolutionseparations


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Commercial CE Chips

Caliper Technologies and Agilent

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Sequencing On-Chip

DNA sequencing on a microchip First demonstrated in 1995 by Mathies group, UCB:

150 bases in 540 s with 97% accuracy In 2002, 96-channel plate demonstrated:

430 bases read in parallel at average rate of 1.7 kb/minwith >99% accuracy

Mathies group


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Integrated Microfluidics

M a t

h i e s e t a l . ,

H i l t o n

H e a

d 2 0 0 2

Mathies group microfabricated 96-channel CE plate with integrated:

Pneumatic valves and pumps ~ PDMS Resistive heaters and temperature

sensors ~ Ti/Pt Photodiode detectors ~ amorphous Si

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PCR On-Chip First on-chip device:

50 L microwell formed in Sisubstrate with anisotropic etching

Bottom of well is SiN membrane withpoly-Si heaters on underside

Cover glass bonded to topsandwiches tubing

Heating rate 15C/sec, cycle time

1 min

Recent developments: Array detection of multiple DNA

fragments Photodiodes integrated in microwells

to detect PCR products byelectrochemiluminescence

Reagent loading with inkjettechnology Gender determination with

CE-PCR, Mathies group

Northrup et al., LLNL Labs


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PCR Devices

Cepheid,Sunnyvale CA

Woolley, Mathies andNorthrup et al., 1996

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Microarrays Fabrication using lithography

and combinatorial chemistry Fodor et al. , 1991

Glass coated with linkermolecule with photoremovableprotective group

UV light through mask removesprotective group selectively

Nucleoside with protected 5

end bonds to deprotectedlinkers Process repeated one base at

a time to give oligonucleotidesof arbitary length

Array of 1024 peptides in 10steps (2 10 ), 100 m probepatches

McGall et al ., 1996, showedtechnique which uses polyimidephotoresist as protective layer

Basic microarray today 50-200 m patches on 1 cm 2 chip

Up to 40,000 different probes Possible oligonucleotides for 15-mer is 4 15

~ 10 9

Finished chip in flow-cell package Detection mainly by fluorescent labeling

Affymetrix process


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How Microarrays Work

S c i e n

t i f i c A m e r

i c a n , F

e b 2 0 0 2

Gene expression

profiling Investigate geneexpression inhealthy anddiseased cellpopulations bymonitoringmessenger RNA incell nuclei.

mRNAs areextracted, reverse-transcribed into

complementaryDNA, andfluorescently labeled

cDNA is hybridizedto microarray.

untreated treated

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Developments Affymetrixs GeneChip

Application-specific microarrays Human set > 33,000 human genes ~ $800/2 chips

Workstation where hybridization, analysis anddata mining are performed

Issues Detection time is diffusion-controlled and slow

Nanogen uses electric fields to direct sample toprobes 25 quicker

By reversing direction of field, can denatureincorrectly bonded strands

Single base pair mismatches (SBPMs) denature 4 faster than exact matches

Need high-density electrically addressable circuitplane

Cheaper detection in the works, i.e. electrochemi-luminescent labeling

Affymetrix

Nanogen

Molecular Probes

Documents

Microfluidics for DNA Analysis