Publication details
Refined quantitative fluorescent PCR of Y-chromosome DNA sequences mosaics in Turner’s syndrome patients – alternative to real-time PCR
| Authors | |
|---|---|
| Year of publication | 2004 |
| Type | Article in Periodical |
| Magazine / Source | Journal of Biochemical and Biophysical Methods |
| MU Faculty or unit | |
| Citation | |
| Doi | http://dx.doi.org/10.1016/j.jbbm.2004.05.004 |
| Field | Genetics and molecular biology |
| Keywords | Capillary electrophoresis; Real-time PCR; Turner’s syndrome; Y-chromosome sequences; QF PCR; FISH; Mosaicism |
| Description | Background: Real-time polymerase chain reactions (PCRs) are the most frequently used techniques for gonosomal mosaics quantification. The primary aim of this work is to assess and optimize the refined technique of quantitative fluorescent polymerase chain reaction (RQF PCR) in the quantification of Y-chromosome sequences in gonosomal mosaics. The method was applied to the analysis of Y-chromosome sequences (amelogenin gene, AMELX/Y-loci) in peripheral lymphocytes and gonadal tissues in Y-positive Turner’s syndrome (TS) patients. Methods: RQF PCR was used for molecular quantification, and fluorescent in situ hybridization (FISH) technique was used for comparison. Results: Based on a formulated calibration curve, DNA mosaics from six Y-positive patients and gonads from one patient were deducted. For calculation of rare mosaics, it is possible to take advantage of a new empirical formula. FISH results were comparable to RQF PCR. Conclusion: The sensitivity of RQF PCR brings significant progress in the analysis of gonosomal aberrations. RQF PCR also finds applications in prenatal diagnostics of maternal contaminations of amniotic fluid and foetal DNA in maternal blood and analysis of chimerism in patients after bone marrow transplantation. The method is very convenient for determining the number of testis-specific protein, Y-linked (TSPY) gene repetitions. |