title: A technique for eliminating allele specific amplification failure during DNA amplification of heterozygous cells for preimplantation diagnosis.
abstract: Advances in techniques of molecular biology have made possible the amplification of specific genes from single cells. This has a major clinical application in preimplantation diagnosis of monogenic disorders. However, the incidence of allele specific amplification failure (allele drop out) in heterozygous single cells can lead to misdiagnosis and the transfer of affected embryos. Few studies have been done to investigate the actual cause of allele drop out, although some investigators have succeeded in reducing but not eliminating it. Here we report the efficiency of amplifying both alleles in heterozygous cells lysed according to two different protocols. A total of 177 heterozygous cells from carriers of cystic fibrosis (CF) and haemoglobin C (HbC) were lysed using two different lysis buffers. Interestingly none of the cells that were lysed with sodium dodecyl sulphate/proteinase K showed any example of allele specific amplification failure whereas in those lysed by KOH/dithiothreitol it was present in 17.6 and 4.7% of the CF and HbC cells respectively. Our results suggest that the phenomenon of allele specific amplification failure is at least in part dependent on the lysis buffer used.
authors.affiliation: Human Genetics Group, The Galton Laboratory, University College London, UK.
authors.forename: N
authors.initials: N
authors.lastname: El-Hashemite
authors.forename: J D
authors.initials: JD
authors.lastname: Delhanty
mesh_terms: D000483:Alleles; D004247:DNA / Q000032:analysis*; D005260:Female; D005307:Fertilization in Vitro; D006579:Heterozygote; D006801:Humans; D008297:Male; D019836:Preimplantation Diagnosis / Q000379:methods*
chemical_list: D004247:DNA