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The use of the Mastercycler gradient for single-cell PCR and preimplantation ,,, diagnosis

Karen Sermon, M.D., PhD
Center for Medical Genetics,
University Hospital and Medical School of the Dutch-speaking Brussels Free University,
Laarbeeklaan 101, 1090 Brussels, Belgium

Introduction

Preimplantation diagnosis (PGD) is a very early form of prenatal diagnosis: embryos obtained in vitro from couples at risk of contracting genetic disease are analyzed for the presence of this disease and only the embryos which are shown to be free of the disease being tested for are transferred to the uterus. The PCR* (Polymerase Chain Reaction) has been the only tool available for diagnosing monogenetic diseases at the DNA level in single cells. At the Center for Medical Genetics, we have developed PGD protocols for numerous diseases, including Cystic Fibrosis (CF) where primarily the DF508 mutation was analyzed, Duchenne's

Materials and Methods

As a source of single cells, lymphoblasts transformed with the Epstein-Barr virus were used. The three cell populations used were from the following individuals; a carrier of the DF508 mutation, a female heterozygous for the CA repeat in intron 45 of the DMD gene, and an individual heterozygous for the DM-repeat (5 and 12 repeats) respectively. The cells were washed three times with PBS, after which individual cells were washed three times in 2 l drops of Ca2+-free and Mg2+-free medium and transferred to 200 l PCR tubes (Eppendorf PCR tubes) containing 2.5 or 5 l alkaline lysis buffer (ALB; 50 mM DTT and 200 mM KOH for CF and DMD or NaOH for DM).

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