1. Multiplex real-time PCR using temperature sensitive primer-supplying hydrogel particles and its application for malaria species identification
- Author
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Sang Jun Sim, Junsun Kim, Sang Kyung Kim, Seungwon Jung, Chae Seung Lim, Changhoon Yoo, Mun Sub Byoun, and Sung Woo Kim
- Subjects
0301 basic medicine ,Plasmodium ,lcsh:Medicine ,Artificial Gene Amplification and Extension ,Polymerase Chain Reaction ,Biochemistry ,law.invention ,chemistry.chemical_compound ,0302 clinical medicine ,Animal Cells ,law ,Nucleic Acids ,Red Blood Cells ,Primer dimer ,Medicine and Health Sciences ,Multiplex ,Particle Spin ,lcsh:Science ,Polymerase chain reaction ,Protozoans ,Multidisciplinary ,Physics ,Temperature ,Malarial Parasites ,Eukaryota ,Hydrogels ,Plasmodium Falciparum ,Real-time polymerase chain reaction ,Physical Sciences ,Temperature sensitive ,Cellular Types ,Research Article ,Materials by Structure ,Amorphous Solids ,Materials Science ,030231 tropical medicine ,Real-Time Polymerase Chain Reaction ,Research and Analysis Methods ,03 medical and health sciences ,Parasitic Diseases ,Animals ,Particle Physics ,Molecular Biology Techniques ,Molecular Biology ,Blood Cells ,Chromatography ,lcsh:R ,Organisms ,Biology and Life Sciences ,Cell Biology ,Tropical Diseases ,Parasitic Protozoans ,Malaria ,030104 developmental biology ,chemistry ,Mixtures ,Nucleic acid ,lcsh:Q ,Primer (molecular biology) ,Multiplex Polymerase Chain Reaction ,Gels ,DNA - 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.
- Published
- 2018