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

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. 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 DNP_ Kit [CitmaGen], 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. 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. 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

[Detection of unknown deletions in alpha globin genes in alpha thalassemia carriers using real-time PCR]

Alpha-thalassemia is one of the most prevalent hemoglobin disorders in the world and it is a common hereditary condition caused by deletion of one or more alpha-globin genes. Common alpha-thalassemia deletions like 3.7 kb, 4.2 kb, 20.5 kb and Med can be detected by Multiplex PCR. There are, however, some unknown deletions that can not be detected by the mentioned method or even by direct DNA sequencing. In the present study, Real-time PCR was used to determine the presence or absence of unknown deletions. Real-time PCR was performed using intercalating dye SYBR Green I and alpha1, alpha2 and CLCN7 genes were amplified. Data analysis was conducted using comparative threshold method [delta delta CT] for determination of Gene dosage of alpha1-globin and alpha2-globin genes. The results showed the ratio of 0.90 +/- 0.16 for normal individuals and the ratio of 0.32 +/- 0.15 for carrier samples with deletions. In addition, Melting curve analysis confirmed the specific amplification of target genes. The Real-time PCR assay is simple, rapid, and reliable. It can be applied for direct determination of unknown deletions in Alpha-thalassemia carriers