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Although prenatal diagnosis has reduced the number of beta-thalassaemia 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 detection of selected beta-thalassaemia mutations in intron I at the single 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 amplification failure after the beta-globin gene amplification from single cells. 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 allows the direct visualization of each allele and provides a simple means of assessing allele-specific amplification failure. Our results show that the combination of nested PCR and automated silver stained SSCP analysis offers exceptional resolution, accuracy and speed which are essential for preimplantation diagnosis.

Original publication




Journal article


Mol Hum Reprod

Publication Date





693 - 698


Alleles, Base Sequence, Blastomeres, Coloring Agents, DNA Primers, Exons, Female, Genetic Carrier Screening, Globins, Homozygote, Humans, Introns, Mutation, Point Mutation, Polymerase Chain Reaction, Polymorphism, Single-Stranded Conformational, Pregnancy, Prenatal Diagnosis, Silver, beta-Thalassemia