SINGLE-CELL DETECTION OF BETA-THALASSEMIA MUTATIONS USING SILVER-STAINED SSCP ANALYSIS - AN APPLICATION FOR PREIMPLANTATION DIAGNOSIS

Although prenatal diagnosis has reduced the number of beta-thalassaemi a births, pregnancy termination is still unacceptable for many couples . Preimplantation genetic diagnosis carried out on the third day after in-vitro fertilization offers an alternative. Here we describe the de tection of selected beta-thalassaemia mutations in intron I at the sin gle cell level by the application of nested polymerase chain reaction (PCR) and silver stained single strand conformation polymorphism (SSCP ) analysis. A total of 294 single somatic cells of different types was amplified with 96% success and all tested mutations in homozygous and heterozygous form were identified correctly. None of the heterozygous or compound heterozygous samples showed any allele-specific amplifica tion failure after the beta-globin gene amplification from single cell s. To assess the efficiency of nested PCR on single blastomeres prior to clinical application, 10 single blastomeres were amplified and gave the expected normal pattern when analysed by SSCP. The main advantage of SSCP, particularly for preimplantation diagnosis, is that it allow s the direct visualization of each allele and provides a simple means of assessing allele-specific amplification failure. Our results show t hat the combination of nested PCR and automated silver stained SSCP an alysis offers exceptional resolution, accuracy and speed which are ess ential for preimplantation diagnosis.