Simultaneous Single-Cell Detection of Two Mutations for Cystic Fibrosis

PURPOSE: A single-cell diagnosis procedure using polymerase chain reaction (PCR) technology was developed to simultaneously detect two cystic fibrosis (CF) mutations (DF-508, W1282X). METHODS: The reported test procedures made use of specific cell lines (lymphoblasts, fibroblasts) of known CF mutation status to determine the efficiency of signal generation and prevalence of allele dropout (ADO) during amplification. RESULTS: Using cells carrying the DF-508 mutation, the PCR signal efficiency for the affected homozygous, normal homozygous, and carrier heterozygote cell populations were 91%, 81%, and 92%, respectively. The total combined PCR efficiency was 87.7% and the ADO rate was 5.7%. For W1282X carrier heterozygote cells, the PCR signal efficiency was 82.0% and the ADO rate was 8.7%. CONCLUSIONS: Methods have been developed to detect two common mutations simultaneously for CF in single-cell assays. The high signal efficiencies and low ADO rates obtained in these tests allow those embryos from couples wishing to avert the transmission of this serious genetic disease to their offspring to be screened by preimplantation genetic diagnosis.