Abstract
Real-time PCR, also called quantitative PCR (qPCR), has been powerful analytical tool for detection of nucleic acids since it developed. Not only for biological research but also for diagnostic needs, qPCR technique requires capacity to detect multiple genes in recent years. Solid phase PCR (SP-PCR) where one or two directional primers are immobilized on solid substrates could analyze multiplex genetic targets. However, conventional SP-PCR was subjected to restriction of application for lack of PCR efficiency and quantitative resolution. Here we introduce an advanced qPCR with primer-incorporated network (PIN). One directional primers are immobilized in the porous hydrogel particle by covalent bond and the other direction of primers are temporarily immobilized at so-called 'Supplimers'. Supplimers released the primers to aqueous phase in the hydrogel at the thermal cycling of PCR. It induced the high PCR efficiency over 92% with high reliability. It reduced the formation of primer dimers and improved the selectivity of qPCR thanks to the strategy of 'right primers supplied to right place only'. By conducting a six-plex qPCR of 30 minutes, we analyzed DNA samples originated from malaria patients and successfully identified malaria species in a single reaction.
Highlights
Real-time PCR has been used for decades as a gold standard for analysing the genes of various diseases.[1,2,3,4] The development of multiplex quantitative PCR (qPCR) systems has been necessary for the quantification of various genetic biomarkers in a single trial
The supplimers with a Tm of 36.0, 45.2, and 55.1 ̊C showed no significant difference in fluorescence intensities after rinsing, but the fluorescence of the supplimeraided primer-immobilized network (sPIN) particle with a supplimer of 18.6 ̊C in Tm clearly decreased compared with the others, which means that the storage of that primer is unstable under room temperature conditions
We demonstrate a new strategy for overcoming the limitations of conventional Solid phase PCR (SP-PCR) by enhancing its efficiency and reliability
Summary
Real-time PCR has been used for decades as a gold standard for analysing the genes of various diseases.[1,2,3,4] The development of multiplex qPCR systems has been necessary for the quantification of various genetic biomarkers in a single trial. The supplimers with a Tm of 36.0, 45.2, and 55.1 ̊C showed no significant difference in fluorescence intensities after rinsing, but the fluorescence of the sPIN particle with a supplimer of 18.6 ̊C in Tm clearly decreased compared with the others, which means that the storage of that primer is unstable under room temperature conditions.
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