Reliability of the single cell PCR for the successful preimplantation genetic diagnosis of single gene disorders

Abstract

Objective: The reliability of single cell PCR analysis is essential for the successful preimplantation genetic diagnosis (PGD) of single gene disorders. Amplification failure and allele drop out (ADO) are considered as the major problem in the single cell PCR. In this study, we evaluated the reliability of duplex nested PCR protocols for single gene disorders using isolated single lymphocyte and blastomere. Design: Laboratory experiment for the clinical application of single cell PCR Materials and Methods: The isolated lymphocytes from carriers of Duchenne muscular dystrophy (DMD), ornithine transcarbamylase (OTC) deficiency and epidermolysis bullosa (EB) and blastomeres from donated abnormal embryos were placed directly into a tube with lysis solution. The single cell was underwent the simultaneous amplification of the causative mutation locus, such as distrophin, OTC and integrin β4. In case of the X-chromosome linked disease (DMD and OTC), SRY gene of Y-chromosome was simultaneously analyzed by the duplex nested PCR for gender selection. The PCR products were confirmed by direct sequencing or RFLP anlysis. Results: Successful amplification rates of distrophin, OTC, integrin β4 gene using single lymphocyte or blastomere were 91.0% (51/56), 91.3% (42/46) and 95.9% (166/173), respectively. Amplification failure occurred in 10.0% (5/50) of SRY gene. ADO rates were in 13.3% (4/30) of OTC and 8.9% (7/78) of integrin β4 gene, respectively. Conclusions: The duplex nested PCR method provided the sufficient reliability (> 90%) in the single cell analysis. This protocol could be applied to clinical PGD cases, successfully. However, we need further studies for optimization of single cell PCR condition to decrease the ADO rate.