Continuous-Flow Microfluidic Device for Real-Time Polymerase Chain Reaction

2016 ◽  
Vol 37 (11) ◽  
pp. 1878-1881
Author(s):  
Hanok Kim ◽  
Shinae Suk ◽  
Kwanseop Lim ◽  
Nokyoung Park ◽  
Jong Hoon Hahn
Author(s):  
Sumeet Kumar ◽  
Todd Thorsen ◽  
Sarit Kumar Das

Polymerase Chain Reaction (PCR) is a molecular biological method for the in vitro amplification of nucleic acid molecules which has wide applications in the area of genetics, medicine and biochemistry. The typical three step PCR cycle consists of heating the sample to 90–94 °C to denature double-stranded DNA, cooling down to 50–54 °C to anneal the specific primers to the single stranded DNA and finally increasing the temperature to 70–75 °C for extension of the primers with thermostable DNA polymerase. The temperature sensitivity of the reaction requires precise temperature control and proper thermal isolation of these three zones. In this paper we present the design of a continuous flow PCR microfluidic device with the channels fabricated in (poly) dimethylsiloxane (PDMS) and thin film Platinum Resistance Temperature Detector (RTD) elements fabricated on glass substrate to define the three different temperature zones. The fluidic arrangement has a water jacket layer to minimize evaporation from the porous PDMS walls. A detailed thermo fluidic model of the device is presented to predict the performance and efficacy of the proposed design. Numerical simulations are carried out to find the temperature distribution and temperature gradients in the device and a parametric study is done by varying flow rate, heat flux and channel dimensions in order to optimize the design for achieving temperature isolation and sharp temperature gradients between different zones.


Author(s):  
Michael B. Sayers ◽  
Tara M. Dalton ◽  
Mark R. Davies

Real-time Polymerase Chain Reaction (PCR) is the preferred method for quantification of gene expression levels due to its extreme sensitivity. Fluorescence based real-time PCR is commonly used for the quantification of the initial amount of a specific sequence of DNA. Real-time quantification may be achieved using fluorescent dyes, by optically monitoring the product formation as the PCR cycles progress. Stationary well based real-time quantification is quite common, however continuous flow real-time PCR which is the aim of this work is still in its infancy. A compact, high throughput continuous flow thermal cycler has been developed which allows for real-time fluorescent measurements to be obtained. The principle of operation of this device is that the three thermal zones required for a polymerase chain reactor are maintained on both sides of an aluminium block and bio-compatible FEP Teflon capillary tubing is then wrapped around these constant temperature blocks. The capillary tubing is wrapped around the device fifteen times which provides thirty PCR thermal cycles. The device has been designed and optimised to accurately monitor the product expression level using the double stranded DNA binding dye SYBR green I. Initially the PCR mixture is segmented into small nanoreactors, separated by an immiscible carrier fluid to eliminate cross contamination and reduce the likelihood of sample degradation due to contact with the capillary wall. These PCR nanoreactors are then cycled through the tubing and the DNA amplified. Fluorescent optical monitoring of these nanoreactors takes place where a water glycerine mixture, which is refractive index matched to the tubing, allows for improved fluorescent measurements of the nano-volume reactors to be obtained. Plasmid DNA, 240 base pairs long, has been successfully amplified using this device and the temperatures for the denaturation, annealing and extension phases have been accurately measured. Real-time fluorecence images of the flowing nano-volumes were recorded every second cycle using a CCD camera and from these images amplification curves have been successfully generated. Samples with various initial concentrations of DNA have been thermally cycled on the continuous flow reactor. The measured increase in fluorescence intensity from the flowing nano-volume reactors as they progressed through the thermal cycler demonstrated the effect of initial DNA template concentration.


The Analyst ◽  
2020 ◽  
Vol 145 (7) ◽  
pp. 2767-2773 ◽  
Author(s):  
Bing Shi ◽  
Yuanming Li ◽  
Di Wu ◽  
Wenming Wu

The polymerase chain reaction (PCR) has unique advantages of sensitivity, specificity and rapidity in pathogen detection, which makes it at the forefront of academia and application in molecular biology diagnosis.


2006 ◽  
Vol 175 (4S) ◽  
pp. 485-486
Author(s):  
Sabarinath B. Nair ◽  
Christodoulos Pipinikas ◽  
Roger Kirby ◽  
Nick Carter ◽  
Christiane Fenske

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