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Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing
L C Waters1, S C Jacobson, N Kroutchinina
1Oak Ridge National Laboratory, Tennessee.
Analytical Chemistry
|February 14, 1998
Summary
This study presents a microchip that integrates cell lysis, multiplex polymerase chain reaction (PCR) amplification, and rapid electrophoretic analysis for DNA. This streamlined process enables quick DNA analysis from biological samples.
Area of Science:
- Biotechnology
- Molecular Biology
- Analytical Chemistry
Background:
- Traditional DNA analysis involves multiple complex steps.
- Microfluidic devices offer potential for integrated biological sample processing.
- Rapid and efficient DNA amplification and separation are crucial for diagnostics.
Purpose of the Study:
- To develop and demonstrate a monolithic microchip for integrated cell lysis, multiplex PCR, and electrophoretic DNA analysis.
- To achieve rapid DNA amplification and size separation on a single device.
- To validate the microchip's performance using specific DNA targets.
Main Methods:
- Sequential execution of cell lysis, multiplex PCR amplification, and electrophoretic analysis on a monolithic microchip.
- Thermal cycling for cell lysis and DNA amplification.
- Size separation using sieving media (hydroxyethyl cellulose or poly(dimethylacrylamide)) and fluorescence detection with an intercalating dye.
- Analysis of amplified DNA fragments from bacteriophage lambda and E. coli.
Main Results:
- Successful integration of cell lysis, multiplex PCR, and electrophoresis on a single microchip.
- Amplification of specific DNA regions (500 bp from bacteriophage lambda; 154, 264, 346, 410, 550 bp from E. coli).
- Electrophoretic analysis completed in under 3 minutes post-amplification.
- Accurate DNA sizing demonstrated using a DNA sizing ladder.
Conclusions:
- The developed microchip enables rapid, integrated DNA analysis.
- This technology has potential applications in molecular diagnostics and high-throughput screening.
- The microchip platform offers a streamlined approach to complex molecular biology workflows.