ANALYSIS OF SEX AND DELTA-F508 IN SINGLE AMNIOCYTES USING PRIMER EXTENSION PREAMPLIFICATION

Reliable sex determination is an inevitable prerequisite in prenatal a nd preimplantation diagnosis of X-linked diseases. We report on an ame logenin-based nested polymerase chain reaction sexing method that simu ltaneously amplifies distinguishable fragments from both sex chromosom es. Primers matching a largely homologous region on both sex chromosom es are used that encompass a 177-bp deletion on the Y chromosome. Thus amplification results in X- and Y-specific fragments of different siz es that are resolved simply by agarose gel electrophoresis. We applied our sexing strategy to 102 single amniocytes previously subjected to primer extension preamplification. 95 showed successful amplification (93.14% sensitivity). The genotyping of all successful amplifications (from 42 male and 53 female amniocytes) was found to be correct (100% specificity). None of the media blanks showed amplification products ( no false positives). Additional amplification of the locus of the most common cystic fibrosis mutation resulted in 95.1% success: 89 amniocy tes (87.3%) showed no mutated allele and 7 (6.9%) were found to be het erozygous for the Delta F508 mutation.