Development and Application of Real-Time Quantitative Polymerase Chain Reaction Technique Using SYBR Green I in the Diagnosis of Down Syndrome

Abstract

Objective: Rapid diagnosis of Trisomy 21 Syndrome (Down Syndrome) patients usingReal-Time quantitative Polymerase Chain Reaction (Real-Time qPCR) in order to establisha novel method for prenatal diagnosis in the future.Materials and Methods: A total of 5 ml of peripheral blood was obtained from eachpatient and normal controls (NR). Then, genomic DNA from lymphocytes was extractedusing the salting out procedure. Gene dosage levels of DSCAM and (PMP22, DSCAM) inDown Syndrome and NR were analyzed using real-time quantitative PCR. The DSCAM/PMP22 ratio was calculated according to the 2-ΔΔCt formula for all samples.Results: Real-time PCR showed a DSCAM/PMP22 ratio of 1.48 ± 0.18 and 1.01 ± 0.10(p<0.001) in Down Syndrome and normal samples, respectively, demonstrating threecopies of the target (DSCAM) gene in Trisomy 21 Syndrome.Conclusion: DSCAM/PMP22 ratio is increased significantly in Down Syndrome patientsthan NR (1.5 times). Therefore, the real-time quantitative PCR technique can be used asa sensitive, accurate and reliable technique for rapid and prenatal diagnosis of Trisomy21 Syndrome.

Keywords

• Down Syndrome
• Trisomy 21
• Gene Copy Number
• Real-Time
• PCR