RESUMO
There is increasing interest in the use of preimplantation genetic diagnosis (PGD) as an alternative to routine prenatal diagnosis. However, the costs associated with development and testing of new PGD protocols have forced some PGD centres to limit the number of diseases for which PGD is offered. One of the main factors in the design of new protocols, which affects cost and accuracy, is the choice of the mutation-detection technique. We have assessed the reliability of DNA sequencing and mini-sequencing for clinical diagnosis at the single-cell level and have found them to be rapid and accurate. Extensive optimisation for individual mutations is not usually necessary when employing these versatile techniques and consequently a smaller investment of time and resources should be required during development of new protocols. Additionally, we report single-cell protocols for the diagnoses of cystic fibrosis, sickle cell anaemia and beta-thalassaemia, which utilise mini-sequencing. Unlike most mutation-detection techniques, mini-sequencing permits analysis of very small DNA fragments. Small amplicons experience low allele dropout (ADO) rates, and consequently this approach could potentially improve the reliability of PGD.