Prospects and Applications of Innovated Quantitative Fluorescent PCR (IQFPCR) in Analyses of Genetic Mosaics Using Gonosomal Sequences

Overview

Background.
Quantification of fluorescence labelled PCR products on capillary electrophoresis (QF PCR) has limitsprimarily in the possibility of more sensitive analyses to detect minority cell lines and small time related variations.PCR efficiency and human factor affect measuring error and reproducibility of results in these cases. The aim of thiswork was to assess and optimise of innovated (I)QF PCR in quantification of Y sequences in gonosomal mosaics.Methods and Results. Artificially prepared Y/X mosaics were tested and quantified by IQF PCR, which replacesreal-time PCR. Comparison of relative fluorescence to PCR cycles in different Y/X dilutions was plotted on thegraphs. Calibration curve for Y sequences quantification was set by the analyses of ratio of Y/X fluorescent signals.An empirical formula was created for the rare mosaic calculation.Conclusions. QF PCR refined by manual real-time PCR eliminates limits of QF PCR and specifies quantitativeanalyses based on PCR. The outstanding feature of IQF PCR is its high sensitivity and accuracy in quantification ofY/X gonosomal mosaics.

Key words:
gonosomal mosaicism, capillary electrophoresis, DNA quantification, real-time PCR, QF PCR.