Allele dropout in sequential PCR and FISH analysis of single cells (cell recycling)

Purpose: Our purpose was to investigate the feasability of using sequential PCR and FISH analysis of single cells for preimplantation diagnosis. Methods: Protocols for sequential PCR and FISH analysis of a single fibroblast (cell recycling) were optimized for six loci and the rates of allele specific dropout (ADO) were determined. Results: Conditions that allow reliable genotyping of single cells in lysis buffer were not optimal for amplifying fibroblasts fixed to coverslips. After optimizing conditions, we observed a success rate of 85% for both analyses in sequential PCR-FISH experiments in single cells for the four loci studied. The individual success rates for each technique revealed a slightly higher rate for FISH (91-95%) than for PCR (85-87%) for single cells on coverslips. The presence of two hybridization signals in FISH experiments demonstrated that the failure to amplify both alleles from heterozygous cells on coverslips was due to true ADO, and not the loss of chromosomal material. The ADO rate observed on cover-slips varied between 10 and 14%, which is significantly higher than that observed in solution, even after meticulous optimization. Conclusions: Sequential PCR and FISH analysis of single cells remains an attractive possibility. However, until the problem of the increased rate of ADO is resolved cell recycling should not be applied to clinical preimplantation genetic analysis.