Preimplantation genetic diagnostic protocols for alpha- and beta-thalassaemias using multiplex fluorescent PCR

Haemoglobinopathies including alpha- and beta -thalassaemia are the world's most common class of single gene disorder. Prenatal diagnosis (PND) for be ta -thalassaemia has been proven to be an effective strategy for controllin g the incidence of new cases and is widely used in several countries where the disease is common. Successful preimplantation genetic diagnosis (PGD) p rotocols for beta -thalassaemia have been introduced using restriction frag ment length polymorphism (RFLP), single-stranded conformation polymorphism. (SSCP) and denaturing gradient gel electrophoresis (DGGE). However, contam ination and allele dropout (ADO) remain an important concern for all of the se strategies. In the present study two PGD protocols for detecting beta -t halassaemia mutations (codon 41-42 and IVSI-110) and one for alpha -thalass aemia (SEA mutation) have been designed and tested. These methods contain f ailsafe mechanisms to reduce the risk of misdiagnosis due to ADO or contami nation and utilise multiplex fluorescent PCR (F-PCR). Interestingly, amplif ication efficiency and ADO were significantly affected by the choice of DNA polymerase and the freshness of the single cells used. The close similarit y between the DNA sequences of beta -globin and delta -globin was also foun d to be an important issue that necessitated careful design of primers for the beta -globin gene. Copyright (C) 2001 John Wiley & Sons, Ltd.