Molecular Human Reproduction, Vol. 9, No. 7, 399-410,
July 2003
© 2003 European Society of Human Reproduction and Embryology
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The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders
Submitted on July 26, 2002; resubmitted on December 24, 2002. accepted on February 27, 2003
1 ‘Genoma’ Molecular Genetics Laboratory, via Po nr. 102, 00198 Rome, 2 S.I.S.Me.R. Reproductive Medicine Unit, via Mazzini nr. 12, 40138 Bologna, 3 Consultorio di Genetica, via Po nr. 45-102, 00198 Rome and 4 EmbryoGen Preimplantation Genetic Diagnosis Centre, via Po nr. 102, 00198 Rome, Italy
5 To whom correspondence should be addressed. e-mail: fiorentino{at}laboratoriogenoma.it
We have applied a new method of genetic analysis, called ‘minisequencing’, to preimplantation genetic diagnosis (PGD) of monogenic disorders from single cells. This method involves computer-assisted mutation analysis, which allows exact base identity determination and computer-assisted visualization of the specific mutation(s), and thus facilitates data interpretation and management. Sequencing of the entire PCR product is unnecessary, yet the same qualitative characteristics of sequence analysis are maintained. The main benefit of the minisequencing strategy is the use of a mutation analysis protocol based on a common procedure, irrespective of the mutations involved. To evaluate the reliability of this method for subsequent application to PGD, we analysed PCR products from 887 blastomeres including 55 PGD cases of different genetic diseases, such as cystic fibrosis, ß-thalassaemia, sickle cell anaemia, haemophilia A, retinoblastoma, and spinal muscular atrophy. Minisequencing was found to be a useful technique in PGD analysis, due to its elevated sensitivity, automation, and easy data interpretation. The method was also efficient, providing interpretable results in 96.5% (856/887) of the blastomeres tested. Fifteen clinical pregnancies resulted from these PGD cases; conventional prenatal diagnosis confirmed all the PGD results, and 10 healthy babies have already been born. Its applicability to PGD could be helpful, particularly in cases in which the mutation(s) involved are difficult to assess by restriction analysis or other commonly used methods.
Key words: allele drop-out/minisequencing/preimplantation genetic diagnosis/single cell PCR/single nucleotide polymorphism
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