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Microfluidics for DNA Analysis

Dr. Thara SrinivasanLecture 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.

• Today’s Lecture• DNA and Analysis Methods • Scaling in Microfluidics• Survey of Microfabricated Chips

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5DNA S

B

P5’ 3’

• Genetic information is stored in chromosomes as long strings of DNA grouped as genes• In humans, 46 chromosomes are 50 - 400 ×106

DNA units long (compared to 4 ×106 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’ ends• 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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5Amplification

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

• Polymerase chain reaction• Double-stranded DNA

denatured, 95°C• Primers attach (anneal) to

strands, flanking section to be amplified, 50-65°C

• Taq enzymes attach to primer sites and synthesize new strands from bases in solution, 72°C

• Repeat cycle 20-30 times to get effective amplification

• Macroscopic thermalcyclers need 90 min per amplification

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Excitation maximumEmission maximum

Detection• Fluorescent labeling with molecules

which emit light when excited allows extremely sensitive visualization of fragment• Intercalating dye: ethidium bromide• Single fluorophore: fluorescein

• Excitation• With UV laser-induced fluorescence,

emission signal must be separated from excitation; requires confocal microscope

• Electrochemiluminescence (ECL) uses Ru(bpy)2+

3 end label, can be detected with conventional CCD

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5Cutting

• Restriction digestion is fragmentation of DNA• Use restriction nuclease enzymes

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

• Size distribution of restriction fragments can fingerprint DNA molecule

Molecular Biology of the Cell

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…and Pasting• Hybridization is hydrogen bonding

of two complementary single strands of DNA• Occurs at specific T and salinity

conditions• In analyses, known strand is probe,

other is unknown and binding indicates match

• Recognition not perfect, single base mismatches occur

• DNA probes immobilized on surface using linker make pixels for microarrays

• Microarray pattern matching

ACGTA

CCGTA GCGTA

TCGTAAGCAT

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5Separation

• Electrophoresis to separate DNA fragments based on size• Mobility µEP 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 = ∆µEP 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 µm in diameter, 50 cm

long• Increased surface to volume ratio and

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

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

fluorescence AgilentTechnologies

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5Sequencing

• Sanger method• Combine PCR and

electrophoretic separations• Duplication of DNA

fragment starts at primer location, as in PCR…

• But in addition to nucleotides in solution, also add small amount ofdideoxy nucleotides (ddNTP’s) of one type (ddA, ddC, ddG, or ddT).

• When ddNTP is captured, growing strand terminates, resulting in…

• …complementary strand fragments terminated at all possible positions for each base

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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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5Today’s Lecture

• DNA and Analysis Methods • Scaling in Microfluidics• Survey of Microfabricated Chips

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Miniaturization Benefits

• Benefits• Reagent consumption ~ [s3]

• Miniscule reaction volumes reduce reagent cost.• Heat transfer ~ [s2]

• Surface phenomena• Mass transfer ~ [s2]

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

• Flow is laminar• Electroosmotic flow for valveless systems ~ [s2] • Capillary flow ~ [s1]

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

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5Miniaturization Issues

• Issues• Detection limit ~ [s3],

• S/N degraded as [s3] unless detector area scales with sample [s1]

• Pressure flows ~ [h3]• Other surface phenomena ~ [s2], [s1]

• Wall adsorption effects and sample evaporation [s2], capillary forces [s1]

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Microfluidics Fabrication

• Fabrication• Batch fabrication

• Microchip cost ~ [s2], but limited by package cost • Parallelization to arrays easy • Portability increased

• Less need for external pumps, detection equipment

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5Scaling 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 x2/2D• For channel width of 70 µm and velocity 1 cm/s,

fluorescein (D = 3 × 10-6 cm2/s) will take 2 s to mix over channel length of 2 mm→ Upper limit of 100 µm width for channels

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5Scaling and Diffusion Effects

• While being carried by electroosmosis and drifted by electrophoresis, a sample slab can spread out in width due to diffusion

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• If initial slab is smaller than Wmin, separation is limited only by diffusion

Ls

U0

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Scaling and Separation Efficiency• Separation efficiency

• Number of theoretical plates, N, per unit time

[ ] ][1,,

,2

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

lLn

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σ

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5Today’s 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 90’s, Manz

group (Imperial College, London) and Harrison group (Univ. of Alberta)

• Separation 100× faster than slab gels, 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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5• Parallelization of CE

using arrays for high throughput• 384-channels radial

microplate for genotyping analyses in <7 min with >98% success

96-channel wafer,

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 Neuchâtel

• Injection schemes to give thinner plug• Higher resolution

separations

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5Commercial 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/min with >99% accuracy

Mathies group

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5Integrated Microfluidics

Mat

hies

et a

l.,

Hilt

on H

ead

2002

• 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 µL microwell formed in Si substrate with anisotropic etching

• Bottom of well is SiN membrane with poly-Si heaters on underside

• Cover glass bonded to top sandwiches tubing

• Heating rate 15°C/sec, cycle time 1 min

• Recent developments:• Array detection of multiple DNA

fragments• Photodiodes integrated in microwells

to detect PCR products by electrochemiluminescence

• Reagent loading with inkjet technology Gender determination with

µCE-PCR, Mathies group

Northrup et al., LLNL Labs

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5PCR Devices

Cepheid,Sunnyvale CA

Woolley, Mathies and Northrup et al., 1996

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Microarrays• Fabrication using lithography

and combinatorial chemistry• Fodor et al., 1991

• Glass coated with linker molecule with photoremovable protective group

• UV light through mask removes protective group selectively

• Nucleoside with protected 5’ end bonds to deprotected linkers

• Process repeated one base at a time to give oligonucleotides of arbitary length

• Array of 1024 peptides in 10 steps (210 ), 100 µm probe patches

• McGall et al., 1996, showed technique which uses polyimidephotoresist as protective layer

• Basic microarray today• 50-200 µm patches on 1 cm2 chip

• Up to 40,000 different probes• Possible oligonucleotides for 15-mer is 415

~ 109

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

Affymetrix process

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How Microarrays Work

Sci

entif

ic A

mer

ican

, Feb

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2

• Gene expression profiling• Investigate gene

expression in healthy and diseased cell populations by monitoring messenger RNA in cell nuclei.

• mRNAs are extracted, reverse-transcribed into complementary DNA, and fluorescently labeled

• cDNA is hybridized to microarray.

untreated treated

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Developments• Affymetrix’s GeneChip

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

• Workstation where hybridization, analysis and data mining are performed

• Issues• Detection time is diffusion-controlled and slow• Nanogen uses electric fields to direct sample to

probes 25× quicker• By reversing direction of field, can denature

incorrectly bonded strands• Single base pair mismatches (SBPMs) denature 4×

faster than exact matches• Need high-density electrically addressable circuit

plane• Cheaper detection in the works, i.e. electrochemi-

luminescent labeling

Affymetrix

Nanogen

Molecular Probes