Interaction of quantitative PCR components with polymeric surfaces.
This study investigated the effect of exposing a polymerase chain reaction (PCR) mixture to capillary tubing of different materials and lengths, at different contact times and flow rates and the adsorption of major reaction components into the tubing wall. Using 0.5 mm ID tubing, lengths of 40 cm and residence times up to 45 min, none of the tested polymeric materials was found to affect subsequent PCR amplification. However, after exposure of the mixture to tubing lengths of 3 m or reduction of sample volume, PCR inhibition occurred, increasing with the volume to length ratio. Different flow velocities did not affect PCR yield. When the adsorption of individual PCR components was studied, significant DNA adsorption and even more significant adsorption of the fluorescent dye Sybr Green I was found. The results indicate that PCR inhibition in polymeric tubing results from adsorption of reaction components to wall surfaces, increasing substantially with tubing length or sample volume reduction, but not with contact time or flow velocities typical in dynamic PCR amplification. The data also highlight that chemical compatibility of polymeric capillaries with DNA dyes should be carefully considered for the design of quantitative microfluidic devices.
Gonzalez
Asensio
A
Stokes Research Institute, University of Limerick, Limerick, Ireland.
Grimes
Ronan
R
Walsh
Edmond J
EJ
Dalton
Tara
T
Davies
Mark
M
D016477:Artifacts; D004247:DNA / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D044085:Microfluidics / Q000295:instrumentation*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011108:Polymers / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D011108:Polymers; D004247:DNA