Continuous-flow DNA and RNA amplification chip combined with laser-induced fluorescence detection

Abstract

DNA and RNA amplification, through polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) are essential steps in most procedures of nucleic acid analysis. Microfabricated devices (chips) for continuous-flow PCR and/or RT-PCR provide very rapid thermal energy transfer and cycling compared to conventional PCR systems. However, time-consuming slab gel electrophoresis has been used for analysis of the amplified fragments obtained from the flow-through chips. In order to facilitate high throughput and automation, we have combined the advantages of continuous-flow DNA and RNA amplification chip with an in-house built laser-induced fluorescence (LIF) detection system that allows analysis of amplification products within seconds. Upon exiting from the chip, the products are mixed with the intercalating dye SYBR Green I (SG1) and introduced into the LIF system. The fluorescence is linearly related to the DNA concentration in the range of 0.025–30 mg/l. The CVs of the LIF measurements range from 0.9 to 12.7%. We studied the effect of the number of amplification cycles on the fluorescence. The fluorescence was also studied as a function of the input DNA molecules in the range of 1.56×10 5–4×10 7. The reproducibility of the entire process including continuous-flow amplification on the chip and LIF detection was 18%. No sample carry-over was observed in the chip or the LIF detector.