TITLE

Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications

AUTHOR(S)
Nakayama, Tsuyoshi; Kurosawa, Yasunori; Furui, Satoshi; Kerman, Kagan; Kobayashi, Masaaki; Rao, S. Ramachandra; Yonezawa, Yuji; Nakano, Kouichi; Hino, Akihiro; Yamamura, Shohei; Takamura, Yuzuru; Tamiya, Eiichi
PUB. DATE
December 2006
SOURCE
Analytical & Bioanalytical Chemistry;Dec2006, Vol. 386 Issue 5, p1327
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (μTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the “initial start-up” in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with μTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.
ACCESSION #
22897038

 

Related Articles

  • Detection of eight GMO maize events by qualitative, multiplex PCR and fluorescence capillary gel electrophoresis. E. Heir; A. Holck // European Food Research & Technology;Jun2008, Vol. 227 Issue 2, p527 

    Abstract  Specific legislation in the EU and several other countries requires that foods containing genetically modified organisms (GMOs) should be approved and labelled. This has necessitated the development of methods for detection of such materials. For screening purposes these methods...

  • Efficient construction of high-density linkage map and its application to QTL analysis in barley. Hori, K.; Kobayashi, T.; Shimizu, A.; Sato, K.; Takeda, K.; Kawasaki, S. // Theoretical & Applied Genetics;Sep2003, Vol. 107 Issue 5, p806 

    Using a High Efficiency Genome Scanning (HEGS) system and recombinant inbred (RI) lines derived from the cross of Russia 6 and H.E.S. 4, a high-density genetic map was constructed in barley. The resulting 1,595.7-cM map encompassed 1,172 loci distributed on the seven linkage groups comprising...

  • PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA, a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants. Swiatek, Magdalena; Greiner, Stephan; Kemp, Sabine; Drescher, Anja; Koop, Hans-Ulrich; Herrmann, Reinhold G.; Maier, Rainer M. // Current Genetics;Jan2003, Vol. 43 Issue 1, p45 

    The genetic transformation of plastids of higher plants has developed into a powerful approach for both basic research and biotechnology. Due to the high copy number of the plastid genome per plastid and per cell, repeated cycles of shoot regeneration under conditions selective for the modified...

  • Detection of Common Transgenic Elements from Soy Sauce Samples by PCRs. Ping Yu; Chunyan Yu // Annual Review & Research in Biology;Oct-Dec2013, Vol. 3 Issue 4, p524 

    Aims: To establish PCR procedures for detecting common transgenic elements from soy sauce samples. Methodology: Soy sauce samples, Haitian, Jiajia, Taitaile and Lijinji, and common transgenic elements from them, the CaMV35S promoter, the NOS terminator, and the Cp4- EPSPS, were chosen to...

  • Applicability of Three Alternative Instruments for Food Authenticity Analysis: GMO Identification. Burrell, A.; Foy, C.; Burns, M. // Biotechnology Research International;2011, p1 

    Ensuring foods are correctly labelled for ingredients derived from genetically modified organisms (GMOs) is an issue facing manufacturers, retailers, and enforcement agencies. DNA approaches for the determination of food authenticitys often use the polymerase chain reaction (PCR), and PCR...

  • Advances in molecular techniques for the detection and quantification of genetically modified organisms. Elenis, Dimitrios S.; Kalogianni, Despina P.; Glynou, Kyriaki; Ioannou, Penelope C.; Christopoulos, Theodore K. // Analytical & Bioanalytical Chemistry;Oct2008, Vol. 392 Issue 3, p347 

    Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO...

  • Expression and production of bioactive human interleukin-18 in transgenic tobacco plants. Bin Zhang; Ying-Hua Yang; Yong-Ming Lin; Qing Rao; Guo-Guang Zheng; Ke-Fu Wu // Biotechnology Letters;Oct2003, Vol. 25 Issue 19, p1629 

    The cDNA of human interleukin-18 (hIL-18) was successfully inserted into the genome of tobacco plant, Nicotiana tabacum cv. NC-89, using Agrobacterium tumefaciens-mediated transformation. Insertion and translation of hIL-18 in transformants were confirmed by PCR, ELISA, and Western blot,...

  • Towards future reference systems for GM analysis. Trapmann, Stefanie; Corbisier, Philippe; Schimmel, Heinz; Emons, Hendrik // Analytical & Bioanalytical Chemistry;Mar2010, Vol. 396 Issue 6, p1969 

    Despite the fact that the measurement unit for the quantification of GMOs in food and feed products has not yet been unambiguously agreed upon in Europe, international trade requires reliable GMO analysis measuring comparably the GMO content of products. The two reference systems, based either...

  • Detection and identification of multiple genetically modified events using DNA insert fingerprinting. Raymond, Philippe; Gendron, Louis; Khalf, Moustafa; Paul, Sylvianne; Dibley, Kim L.; Bhat, Somanath; Xie, Vicki R. D.; Partis, Lina; Moreau, Marie-Eve; Dollard, Cheryl; Coté, Marie-José; Laberge, Serge; Emslie, Kerry R. // Analytical & Bioanalytical Chemistry;Mar2010, Vol. 396 Issue 6, p2091 

    Current screening and event-specific polymerase chain reaction (PCR) assays for the detection and identification of genetically modified organisms (GMOs) in samples of unknown composition or for the detection of non-regulated GMOs have limitations, and alternative approaches are required. A...

Share

Read the Article

Courtesy of THE LIBRARY OF VIRGINIA

Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics