Prospects and Applications of Innovated Quantitative Fluorescent PCR (IQF
PCR) in Analyses of Genetic Mosaics Using Gonosomal Sequences
Vodička R., Vrtěl R., 1Dušek L. , Švábová D. , Šantavý J.
Ústav lékařské genetiky a fetální medicíny LF UP a FN, Olomouc 1Centrum biostatistiky a analýz LF a PřF MU, Brno
Background. Quantification of fluorescence labelled PCR products on capillary electrophoresis (QF PCR) has limits
primarily 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 this
work 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 replaces
real-time PCR. Comparison of relative fluorescence to PCR cycles in different Y/X dilutions was plotted on the
graphs. 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 quantitative
analyses based on PCR. The outstanding feature of IQF PCR is its high sensitivity and accuracy in quantification of
Y/X gonosomal mosaics.
gonosomal mosaicism, capillary electrophoresis, DNA quantification, real-time PCR, QF PCR.