PCR Microfluidics
MICRODroplets
Belayneh Getahun BekeleMaster Program in Nanoscience, Materials and Processes: Chemical Technology at the Frontier
1. Introduction
2. Motivations for PCR Microdevice: Miniaturization
3. Design of the Methods
4. PCR in Microdroplets
5. Detection Methods5. Detection Methods
6. Current Developments: Examples
7. Summary
8. References
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1) INTRODUCTION
Microfluidics refers to the behavior and control of liquids constrained to volumes near the μL range.
Microfluidic Devices
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Giouroudi, I., and Keplinger, F. Int. J. Mol. Sci. 2013, 14, 18535-18556
Microfluidic Devices
Comparisons
Conventional PCR Microfluidic PCR
the thermal mass is large,
which leads to lengthy PCR reactions
Offline sample preparation
Low thermal mass
Rapid heat transfer
Integration of
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Offline sample preparation
Consumption of expensive reagent
Short, chimeric products
Integration of
sample preparation,
separation
amplification
detection,
On a single chip
Less reagentLiu, C.,et al. Anal. Chem. 2006, 78, 5474-5479
2. Motivation for PCR Microdevice: Miniaturization
Low High
Cost of fabrication and use,
analysis time
portability,
integration,
parallel amplification,
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analysis time parallel amplification,
acceptable disposal
Zhang, C., et al. Biotechnology Advances 2006, 24, 243–284
3) DESIGN OF THE METHODS
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Link, D. , et al. Lab Chip 2011,11, 2156−2166
PCR fluid in a microsystem of a chip
MicroPCR devices
well-based PCR chips continuous-flow PCR chips
the PCR mixture in well
the whole chip is heated and cooled.
moves the sample through fixed temperature zones
smaller thermal inertia
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and cooled.
Higher thermal inertia and time
Auroux, P., et al. Lab Chip 2004, 4,534–546 Cao, Q., et al. PLoS ONE 2012 , 7, e33176
Continuous-flow PCR
(A) Serpentine channel (B) Spiral channel-based (C) the circular channel-based
Zhang, C., and Xing, D. Nucl. Acids Res. 2007,35, 4223-4237
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Microfluidic PCR: Limitations
1) interactions between the surface
PCR Inhibition and Carry-over
Limitations similar to Conventional PCR
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2)Preference for short segmentschimeric products
Zhang, Y., and Ozdemi, P. Analytica Chmica Acta 2009, 638, 115-125
Limitations similar to Conventional PCR
4) PCR IN MICRODROPLETS
In vitro compartmentalisation of reactions in water-in-oil droplets
(as in emulsion PCR for sequencing)
reduces recombination between homologous fragments:
Avoids synthesis of short, chimeric products.
Assay carry-over and inhibition is minimized Assay carry-over and inhibition is minimized
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Zhang, Y., and Ozdemi, P. Analytica Chmica Acta 2009, 638, 115-125
6.DETECTION METHODS
TheAnalytical performance,
Real-time vs end point detection
Zhu,Y. , and Fang, Q. Analytica Chimica Acta 2013, 787, 24– 35
Analytical performance,Available probesof each analytical technique
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DNA extractionIn-situ Analyzer
Schematic view of the microfluidicstructure for continuous DNAextraction.
7. CURRENT DEVELOPMENTS: EXAMPLES
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Karle, M., et al. Lab Chip 2010, 10, 3284–3290
Cell lysis,DNA purification, PCR,
and optical detection
In-situ Analyzer
Microfluidic Device for IISA-Gene.
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Schematic illustration
PCR, and optical detection
Fukuba,T.,et al, RSC Adv. 2011,1, 1567-1573
8. SUMMARY
Microfluidic PCR is a better alternative to conventional PCR
Droplet-based Microfluidics is shown to allievate some problems of Microfluidic PCR.
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9) REFERENCES
1. Auroux, P., et al. Lab Chip 2004, 4, 534–546
2. Cao, Q., et al. PLoS ONE 2012 , 7, e33176
3. Fukuba,T,. et al, RSC Adv. 2011,1, 1567-1573
4. Giouroudi, I., and Keplinger, F. Int. J. Mol. Sci. 2013, 14, 18535-18556
5. Hataoka, Y., et al. Anal Sci 2005, 21, 53–56
6. Karle, M., et al. Lab Chip 2010, 10, 3284–3290
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6. Karle, M., et al. Lab Chip 2010, 10, 3284–3290
7. Link, D., et al. Lab Chip 2011,11, 2156−2166
8. Liu, C., et al. Anal. Chem. 2006, 78, 5474-5479
9. Zhang, C., and Xing, D. Nucl. Acids Res. 2007,35, 4223-4237
10. Zhu,Y. , and Fang, Q. Analytica Chimica Acta 2013, 787, 2435
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