The influence of amplicon length on real-time PCR results
(2017) Biotechnologie, Agronomie, Société et Environnement — Vol. 27, n° 1, p. 1-9 (2017)
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- Abstract
- Description of the subject. This paper discusses the influence of amplicon length on real-time PCR results. Objectives. The aim of the experiments was to show that amplicon size has an influence on detection. Method. Tests were performed on genomic and plasmid DNA. Double-dye probes and SYBR® Green were used for detection by real-time PCR. Primers were selected in order to produce fragments with increasing sizes. Experiments dealt with two targets: an endogenous target for soybean (part of the lectin gene) and a transgenic target (junction P35S-CTP of the MON40- 3-2 soybean). Results. The results show that the kinetics of amplification curves evolve as a function of amplicon length, and smaller amplicons yield a higher level of fluorescence for the plateau phase. DNA degradation within the sample as well as the principles of fluorescence acquisition as a function of the chemistry used can also be factors. Conclusions. It was experimentally shown that the observed effect is linked to the suboptimal elongation temperature used in real-time PCR. Detection using SYBR® Green is less impacted as the loss of efficiency is partially compensated by the greater integration of SYBR® Green molecules in the larger fragments.
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Debode, F., Marien, A., Janssen, E., Bragard, C., & Berben, G. (2017). The influence of amplicon length on real-time PCR results. Biotechnologie, Agronomie, Société et Environnement, 27(1), 1-9. https://hdl.handle.net/2078.5/255757 (Original work published 2017)