TY - JOUR
TI - A widely applicable strategy for single cell genotyping of
beta-thalassaemia mutations using DGGE analysis: Application to
preimplantation genetic diagnosis
AU - Vrettou, C
AU - Palmer, G
AU - Kanavakis, E
AU - Tzetis, M
AU - Antoniadi, T
AU - and Mastrominas, M
AU - Traeger-Synodinos, J
JO - Prenatal Diagnosis
PY - 1999
VL - 19
TODO - 13
SP - 1209-1216
PB - Wiley
SN - 0197-3851, 1097-0223
TODO - 10.1002/(SICI)1097-0223(199912)19:13<1209::AID-PD722>3.0.CO;2-6
TODO - preimplantation genetic diagnosis; beta-thalassaemia; PCR amplification;
DGGE analysis
TODO - Preimplantation genetic diagnosis (PGD) allows the selection of
unaffected IVF embryos for transfer in couples that are at risk for
transmitting genetic diseases. For monogenic diseases, polymerase chain
reaction (PCR)-based diagnosis is usually performed on single
blastomeres. In Greece, up to 10 per cent of the population are carriers
for beta-thalassaemia and related haemoglobinopathies, and more than 20
pathological mutations in the beta-globin gene have been described. In
this study we report a strategy which includes a first round of PCR,
allowing subsequent nested PCR and DGGE analysis for at least 95 per
cent of beta-thalassaemia major genotypes in the Greek population. The
use of DCGE for beta-globin genotype analysis is advantageous: it
facilitates simultaneous analysis of more than one mutation in a single
PCR fragment, it detects the presence of normal alleles and monitors the
occurrence of allelic drop-out (ADO) through the expectation that
heterozygous samples have more than one electrophoretic band on DGGE
analysis. The optimization, accuracy and reliability of the method was
evaluated by genotyping 325 single blastomeres, 110 amniocytes and 55
lymphocytes. Results confirmed that PCR efficiency and occurrence of ADO
are improved by higher denaturation temperatures in the first cycles of
first-round PCR, influenced by the size of the fragment amplified in the
first round of PCR and additionally by the quality and type of cells
being genotyped. The proposed strategy was accurate and reliable, and
thus for application to PGD should ensure the transfer of unaffected
embryos. Furthermore it is widely applicable in most of the populations
worldwide where beta-thalassaemia is common. Copyright (C) 1999 John
Wiley & Sons, Ltd.
ER -