Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene

A sensitive technique for the sexing of bovine embryos was developed using polymerase chain reaction (PCR) amplification of the bovine amelogenin (bAM L) gene on the X- and Y-chromosomes of Holstein dairy cattle. Cloning and D NA sequencing showed a 45.1% homology between the fifth intron of the bAML- X and bAML-Y gene with multiple deletions. A pair of sex-specific primers w as designed to allow amplification of a single fragment of 467-bp from the X-chromosome of female cattle and two fragments of 467-bp and 341-bp from t he X- and Y-chromosomes of male cattle. The primers were successfully appli ed to bovine sexing from single blastomeres isolated from day-6 to day-7 co w embryos by direct cell lysis and PCR. Our protocol of embryo sexing shoul d be applicable to the diagnosis of defective genes in vitro in human embry os and in other domestic or recreational animals. (C) 1999 Wiley-Liss, Inc.