Abstract
In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.
Highlights
The polymerase chain reaction (PCR) is the most ubiquitous molecular biology tool in use
SYTO 82 (S82) belongs to the group of SYTO orange dyes which forms, along with the green, blue, and red groups, a broad family of cell-permeant nucleic acid stains
The results proved that the 0.8μM dye: 0.4μM probe ratio sufficiently compensated for the FAM signal loss and improved the emitted FAM signal intensity, yielding consistently steeper amplification curves while keeping the remaining reaction parameters unaffected
Summary
The polymerase chain reaction (PCR) is the most ubiquitous molecular biology tool in use. We investigated how different S82 concentrations affect the basic TaqMan reaction parameters including the FAM fluorescence and sensitivity of the melting analysis.
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