ArticleMikrochimica acta2014
Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency.
Article in Mikrochimica acta, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 43 papers.
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43 citing papers in PubMed, 76 citations in OpenAlex.
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- Use and Misuse of CLife (Basel, Switzerland) · 2021Review
- PANDAA intentionally violates conventional qPCR design to enable durable, mismatch-agnostic detection of highly polymorphic pathogens.Communications biology · 2021Article
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- Intraspecific genetic variability and diurnal activity affect environmental DNA detection in Japanese eel.PloS one · 2021Article
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Authors and funding
5 authors at 4 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The analysis of quantitative PCR data usually does not take into account the fact that the increase in fluorescence depends on the monitoring chemistry, the input of ds-DNA or ss-cDNA, and the directionality of the targeting of probes or primers. The monitoring chemistries currently available can be categorized into six groups: (A) DNA-binding dyes; (B) hybridization probes; (C) hydrolysis probes; (D) LUX primers; (E) hairpin primers; and (F) the QZyme system. We have determined the kinetics of the increase in fluorescence for each of these groups with respect to the input of both ds-DNA and ss-cDNA. For the latter, we also evaluated mRNA and cDNA targeting probes or primers. This analysis revealed three situations. Hydrolysis probes and LUX primers, compared to DNA-binding dyes, do not require a correction of the observed quantification cycle. Hybridization probes and hairpin primers require a correction of -1 cycle (dubbed C-lag), while the QZyme system requires the C-lag correction and an efficiency-dependent C-shift correction. A PCR efficiency value can be derived from the relative increase in fluorescence in the exponential phase of the amplification curve for all monitoring chemistries. In case of hydrolysis probes, LUX primers and hairpin primers, however, this should be performed after cycle 12, and for the QZyme system after cycle 19, to keep the overestimation of the PCR efficiency below 0.5 %. FigureThe qPCR monitoring chemistries form six groups with distinct fluorescence kinetics. The displacement of the amplification curve depends on the chemistry, DNA input and probe-targeting. The observed shift in C
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