[Prospects and applications of innovated quantitative fluorescent PCR (IQF PCR) in analyses of genetic mosaics using gonosomal sequences].
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.


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.
Ustav, lékarské genetiky a fetální medicíny LF UP a FN, Olomouc. vodickar@fnol.cz
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R
Vodicka
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Vrtel
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L
Dusek
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D
Svábová
J
J
Santavý
D002460:Cell Line; D041321:Chromosomes, Human, X; D041322:Chromosomes, Human, Y; D019075:Electrophoresis, Capillary; D005260:Female; D006801:Humans; D008297:Male; D009030:Mosaicism; D016133:Polymerase Chain Reaction