PCR and Microfluidic Integration

Polymerase Chain Reaction (PCR), has been used since the late 1980’s as a normal procedure in molecular biology labs. It consists of the amplification of DNA fragments by a heat stable DNA polymerase an enzyme capable to join nucleotides (ATP, GTP, CTP) as a copy of the original DNA fragment. In these way many DNA copies are obtained; for that reason, PCR is recognized as an amplification reaction. However, long reaction times and multi-step procedures are a disadvantage, for that reason the emerging miniaturized PCR devices will become in a standard tool for molecular diagnosis this will be part of bigger lab-on-a-chip systems (LOC) ⁵. PCR allows the detection of target trace DNA which is relevant for disease diagnosis or gene detection during molecular biology procedures.

Why should you integrate PCR and microfluidics?

For many scientist is very important to reduce the PCR reaction times and generates new ways to take advantage of this reaction, the experimentation on this new system requires peristaltic pumps or syringe pumps, before the PCR-chips comes into play, Yamane group in Japan designed a flow PCR reaction at constant flow using a thin Teflon capillary. The amplification was about 50% than the obtained with conventional thermocyclers. At that time they observed a the elongation time of 24 s which approaches to the theoretical limit for replication of an I kbp DNA fragment ³.

Typical PCR reaction takes hours, to overcome this micro-PCR can reduce the time reaction to a few minutes. In a continuous-flow device which typically conveys three zones at constant temperatures. The three zones cover the denaturation (201-205°F), annealing(154°F) and elongation (161°F)classical steps. The presented micro-PCR methods can amplify DNA between 5 and 10 min. Every cycle is between 10 and 20 seconds. It is possible to produce 86 ng of DNA with a flow of 2.5 µl/min. Considering that the initial amount of template of 1 to 10 ng was used during the experiments, it is possible to get 8 times more product in less time. However, the classical amplification provides with 300 to 400 ng ². Electronic syringe pumps like the ones offered by Chemyx may allow the development of more efficient micro-PCR reactions.

New detection schemes with PCR and microfluidics?

The PCR may allow more sensitive detection methods when other technologies are used, the reaction can be coupled with ligase for flow-through hybridization assay using chemiluminescence. Package probe DNA-immobilized magnetic beads provided accelerated detection of target DNA. The flow was applied with a Chemyx syringe pump system, the preliminary results demonstrated that this analytical system could detect both homozygous and heterozygous mutations, without the expensive instrumentation and cumbersome procedures required by conventional DNA microarray-based methods ¹. This is another example of how Chemyx electronic syringe pumps may help you to save money and allow you the design of new exciting PCR technologies.

Other PCR systems?

Quantitative PCR process is also enhanced by the microfluidic operation; new technologies can process up to 40 qPCR cycles in 20 minutes vs. 1 hour with conventional systems < already proofed the feasibility of using real-time PCR as a diagnostic tool for Rapid Detection of Influenza A/H1N1 virus in human clinical Specimens. They found that is possible to do ultra-fast PCR as a point-of-care-potential diagnostic tool for influenza A/H1N1 with high sensitivity and specificity.

Concluding Remarks

The implementation of microfluidics with PCR allows faster reaction that conventional heat blocks. Also, the integration of these new detection PCR methods and chemiluminescence is possible with microfluidics. Considering the previous review to achieve the microfluidic regime flow, electronic microfluidic syringe pumps are a necessity. Chemyx syringe pump systems can quickly reach the flow which for these specialized systems.

References

1. HOMMATSU, M., OKAHASHI, H., OHTA, K., TAMAI, Y., TSUKAGOSHI, K., HASHIMOTO, M., 2013. Development of a PCR/LDR/Flow-Through Hybridization Assay Using a Capillary Tube, Probe DNA-Immobilized Magnetic Beads and Chemiluminescence Detection. Anal. Sci. 29, 689–695. https://doi.org/10.2116/analsci.29.689
2. Moschou, D., Vourdas, N., Kokkoris, G., Papadakis, G., Parthenios, J., Chatzandroulis, S., Tserepi, A., 2014. All-plastic, low-power, disposable, continuous-flow PCR chip with integrated microheaters for rapid DNA amplification. Sensors Actuators, B Chem. 199, 470–478. https://doi.org/10.1016/j.snb.2014.04.007
3. Nakano, H., Matsuda, K., Yohda, M., Nagamune, T., Endo, I., Yamane, T., 1994. High speed polymerase chain reaction in constant flow. Biosci. Biotechnol. Biochem. 58, 349–352. https://doi.org/10.1080/bbb.58.349
4. Song, H.-O., Kim, J.-H., Ryu, H.-S., Lee, D.-H., Kim, S.-J., Kim, D.-J., Suh, I.B., Choi, D.Y., In, K.-H., Kim, S.-W., Park, H., 2012. Polymeric LabChip Real-Time PCR as a Point-of-Care-Potential Diagnostic Tool for Rapid Detection of Influenza A/H1N1 Virus in Human Clinical Specimens. PLoS One 7, e53325. https://doi.org/10.1371/journal.pone.0053325
5. Verdoy, D., Barrenetxea, Z., Berganzo, J., Agirregabiria, M., Ruano-López, J.M., Marimón, J.M., Olabarría, G., 2012. A novel Real Time micro PCR based Point-of-Care device for Salmonella detection in human clinical samples. Biosens. Bioelectron. 32, 259–265. https://doi.org/10.1016/j.bios.2011.12.032