Use of Real-Time PCR method on the amniocyte samples for prenatal diagnosis of Down syndrome

Objective: In this study, the possibility of prenatal diagnosis of Down syndrome with Real-Time PCR method was evaluated. In this context, optimization of a suitable method for purification of high quality DNA from amniotic fluid samples was also considered.
Materials and Methods: Pregnant women who had the high risk of having babies with Down syndrome were selected according to the biochemical and sonographic data and referred to the amniocentesis center. The DNA of total 59 amniotic fluid samples were extracted with different methods including boiling method, salting out method, Procedures of DNA extraction from Blood and Cell Culture by DNPTM Kit (CinnaGen), Procedure of DNA extraction from cells by DNA Isolation Kit for cells and tissues (Roche), Procedure of DNA extraction from Tissue by MagNa Pure DNA Isolation kit (Roche), and QIAamp DNA Micro Kit (Qiagen). Then, the quality and quantity of the extracted DNA were evaluated by the NanoDrop® ND- 1000 spectrophotometer device. Real-Time PCR reaction using fluorescent dye SYBR Green I (Applied Biosystems, UK) was performed to specifically amplify DSCAM and DYRK1A2 genes and the reference gene (PMP22). Data analysis was performed using comparative cycle threshold method for the determination of the gene dosage and determining the number of copies of chromosome 21.
Results: This study showed that DNA extracted from amniotic fluid samples using QIAamp DNA Micro Kit (Qiagen) has the desirable quantity and quality for Real-Time PCR. Specific proliferation of targets and reference genes was achieved and difference between normal and affected groups based on differences between their gene dosages was determined.
Conclusion: Prenatal diagnosis of Down syndrome is feasible by the Real-Time PCR method using DNA samples from amniotic fluid cells extracted by QIAamp DNA Micro Kit (Qiagen). The results are comparable to the corresponding results from conventional cytogenetic methods.