Microchip based PCR

Lou, X.J., Panaro, N., Wilding, P., Fortina, P., Kricka, L.J., Increased amplification efficiency of microchip-based PCR by dynamic surface passivation. Biotechniques 2004, 36, 248-250. 

Since the early demonstrations of microchip-based PCR, numerous authors have reviewed the topic and forecast the... 

The polymerase chain reaction (PCR) is the most widely used technique for oligonucleotide synthesis. Microchips/microdevices for PCR are studied extensively, and thus great progress has been made in the development and scope of microchip components of microchip-based PCR analyzers such as on-chip micromachining (fabrication, bonding, and sealing), choice of... 

Munro, N.J., Hiihmer, A.F.R., Landers, J.P., Robust polymeric microchannel coating for microchip-based analysis of neat PCR products. Anal. Chem. 2001, 73, 1784-1794. 

Fortina P, Cheng J, Kricka LJ, Waters LC, Jacobson SC, Wilding P. DOP-PCR amplification of whole genomic DNA and microchip-based capillary electrophoresis. Methods in Molecular Biology 2001 163 211-9. 

Munro, N. J. Huhmer, A. F. R. Landers, J. P. Robust Polymeric MicroChannel Coatings for Microchip-Based Analysis of Neat PCR Products. Anal. Chem. 2001,73, 1784-1794... 

In a more direct translation of current macroscale, silica-based, SPE protocols other, microchip-based purification systems have focused on utilizing a packed silica-bead bed or silica sol-gel matrix solid phase for purification. This type of extraction was first miniaturized in a capillary format, to demonstrate the utility of the proposed method in the microscale, by Tian et al., who utilized a 500 nL capillary-based chamber packed with silica particles to establish that PCR-amplifiable DNA (with 80-90% of proteins removed during the load and wash steps) could be obtained from white blood cells with high extraction efficiencies (70%). This demonstrated the feasibility of incorporating such silica-based column purification methods into microfabricated devices and the effectiveness of such methods for the purification of DNA from a wide variety of biological species (white blood cells, cultured cells, and whole blood). 

Lee JG, Cheong KH, Huh N, Choi JW, Ko C (2006) Microchip-based [Pg.2692]

Lee J-G, Cheong KH, Huh N, et al. Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification. Lab Chip. 2006 886-95. doi 10.1039/ B515876A. 

Single-nucleotide polymorphisms (SNPs) have been analysed on a capillary gel electrophoresis (CGE) microchip with EC detection [150]. The genetic section that contained the SNP was amplified by PCR and purified. Then, it was used in a single-base extension (SBE) reaction with a redox-labeled chain terminator, ferrocene-acycloATP. Products of the SBE, ferrocene-labeled SNP and free ferrocene-acycloATP, were separated employing CGE on microchip and detected using sinusoidal voltammetric detection at a pyrolysed photoresist film (PPF) electrode. 

Zhou et al. [175] described the determination of severe acute respiratory syndrome (SARS) coronavirus by a microfluidic chip system. The unit included an LIF microfluidic chip analyzer, a glass microchip for both PCR and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. According to the authors, the system allowed efficient DNA amplification of the SARS coronavirus followed by electrophoretic sizing and detection on the same chip. 

With the need to provide PCR-amplifiable DNA, multiple approaches for incorporation of the extraction protocol onto microchips were examined. Recent development includes the implementation of a solid-phase extraction of DNA on a microchip [49]. The extraction procedure utilized was based on adsorption of the DNA onto bare silica. The silica beads were immobilized into the channel using a sol-gel network. This method made possible the extraction and elution of DNA in a pressure-driven system. 

The polymerase chain reaction is the prevalent method for DNA amplification. Much effort has been made to integrate PCR chambers on microchips to carry out amplifications of DNA molecules prior to their analysis. For instance, PCR was first achieved on a Si-based reaction chamber (25 or 50 pL) integrated with a polysilicon thin-film (2500-A-thick) heater for the amplification of the GAG gene sequence (142 bp) of HIV (cloned in bacteriophage M13) [997]. 

Ibrahim, M.S., Lofts, R.S., Jahrling, P.B., Henchal, E.A., Weedn, V.W., Northrup, M.A., Belgrader, P., Real-time microchip PCR for detecting single-base differences in viral and human DNA. Anal. Chem, 1998, 70(9), 2013-2017. 

Several examples of work in this field have been reported for multiple-sample polymerase chain reaction (PCR) amplifications (464 166) and electrophoretic analysis in a microchip. In one example, the PCR products from four DNA samples were analyzed by microchip gel electrophoresis (466). The ability to analyze a large number of DNA samples in this format was presented. Run-to-run reproducibility in the sizing microchip was very good percent relative standard deviation n = 8 for migration times was better than 0.3%. The accuracy of the 500-base-pair sizing was greater than 98% (466). 

---