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Clin Chim Acta. 2015 Jan 15;439:231-50. doi: 10.1016/j.cca.2014.10.017. Epub 2014 Oct 22.

Real-time PCR detection chemistry.

Author information

1
Research Unit, General University Hospital, Laurel s/n, 02006 Albacete, Spain. Electronic address: enavarro66@yahoo.es.
2
Research Unit, General University Hospital, Laurel s/n, 02006 Albacete, Spain. Electronic address: gemmas@sescam.jccm.es.
3
Research Unit, General University Hospital, Laurel s/n, 02006 Albacete, Spain. Electronic address: castanoaroca@yahoo.es.
4
Internal Medicine Department, General University Hospital, Hermanos Falcó 37, 02006 Albacete, Spain. Electronic address: solera53@yahoo.es.

Abstract

Real-time PCR is the method of choice in many laboratories for diagnostic and food applications. This technology merges the polymerase chain reaction chemistry with the use of fluorescent reporter molecules in order to monitor the production of amplification products during each cycle of the PCR reaction. Thus, the combination of excellent sensitivity and specificity, reproducible data, low contamination risk and reduced hand-on time, which make it a post-PCR analysis unnecessary, has made real-time PCR technology an appealing alternative to conventional PCR. The present paper attempts to provide a rigorous overview of fluorescent-based methods for nucleic acid analysis in real-time PCR described in the literature so far. Herein, different real-time PCR chemistries have been classified into two main groups; the first group comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore-labeled oligonucleotides. The latter, in turn, has been divided into three subgroups according to the type of fluorescent molecules used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor, LUX, Cyclicons, Angler); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or displacing); and (iii) analogues of nucleic acids (PNA, LNA, ZNA, non-natural bases: Plexor primer, Tiny-Molecular Beacon). In addition, structures, mechanisms of action, advantages and applications of such real-time PCR probes and analogues are depicted in this review.

KEYWORDS:

DNA binding dye; DNA detection chemistries; Fluorescent primer-probe; Fluorescent probe; Nucleic acid analogues; Real-time PCR

PMID:
25451956
DOI:
10.1016/j.cca.2014.10.017
[Indexed for MEDLINE]

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