Molecular Human Reproduction, Vol 3, 693-698, Copyright © 1997 by Oxford University Press
N el-Hashemite, D Wells and JD Delhanty
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.
JOURNAL ARTICLE
Single cell detection of beta-thalassaemia mutations using silver stained SSCP analysis: an application for preimplantation diagnosis
Galton Laboratory, University College London, UK.
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