A technique for eliminating allele specific amplification failure during DNA amplification of heterozygous cells for preimplantation diagnosis

Advances in techniques of molecular biology have made possible theamplification of specific genes from single cells. This has a majorclinical application in preimplantation diagnosis of monogenic disorders.However, the incidence of allele specific amplification failure (alleledrop out) in heterozygous single cells can lead to misdiagnosis and thetransfer of affected embryos. Few studies have been done to investigate theactual cause of allele drop out, although some investigators have succeededin reducing but not eliminating it. Here we report the efficiency ofamplifying both alleles in heterozygous cells lysed according to twodifferent protocols. A total of 177 heterozygous cells from carriers ofcystic fibrosis (CF) and haemoglobin C (HbC) were lysed using two differentlysis buffers. Interestingly none of the cells that were lysed with sodiumdodecyl sulphate/proteinase K showed any example of allele specificamplification failure whereas in those lysed by KOH/dithiothreitol it waspresent in 17.6 and 4.7% of the CF and HbC cells respectively. Our resultssuggest that the phenomenon of allele specific amplification failure is atleast in part dependent on the lysis buffer used.