ALLELE DROPOUT IN SEQUENTIAL PCR AND FISH ANALYSIS OF SINGLE CELLS (CELL RECYCLING)

Purpose: Our purpose was to investigate the feasability of using seque ntial PCR and FISH analysis of single cells for preimplantation diagno sis. Methods: Protocols for sequential PCR and FISH analysis of a sing le fibroblast (cell recycling) were optimized for six loci and the rat es of allele specific dropout (ADO) were determined. Results: Conditio ns that allow reliable genotyping of single cells in lysis buffer were not optimal for amplifying fibroblasts fixed to coverslips. After opt imizing conditions, we observed a success rate of 85% for both analyse s in sequential PCR-FISH experiments in single cells for the four loci studied. The individual success rates for each technique revealed a s lightly higher rate for FISH (91-95%) than for PCR (85-87%) for single cells on coverslips. The presence of two hybridization signals in FIS H experiments demonstrated that the failure to amplify both alleles fr om heterozygous cells on coverslips was due to true ADO, and not the l oss of chromosomal material. The ADO rate observed on cover-slips vari ed between 10 and 14%, which is significantly higher than that observe d in solution, even after meticulous optimization. Conclusions: Sequen tial PCR and FISH analysis of single cells remains an attractive possi bility. However, until the problem of the increased rate of ADO is res olved cell recycling should not be applied to clinical preimplantation genetic analysis.