Real-time PCR using molecular beacons for accurate detection of the Y chromosome in single human blastomeres

We describe a highly accurate method for determining the sex of human embry os via real-time polymerase chain reaction (PCR) amplification of highly-co nserved, moderately-repeated sequences within the TSPY genes on the Y chrom osome and the U2 genes on chromosome 17. Individual male lymphocytes, femal e lymphocytes, and blastomeres from donated cleavage-stage embryos were lys ed prior to PCR using an optimized buffer containing proteinase K. Molecula r beacons, a new type of fluorescent probe, were used to detect and quantif y accumulating amplicons during each cycle of PCR carried out in closed tub es. The present work is part of an ongoing study to construct and implement a new, convenient and reliable system of preimplantation genetic diagnosis (PGD).