Sexing of Bovine Embryos with Male-Specific Repetitive DNA by Polymerase Chain Reaction: Cloning and Characterization of BovineMale-Specific Repetitive DNA

概要

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Bovine male-specific repetitive DNAs were cloned for the sexing of bovine embryos by means of the polymerase chain reaction (PCR). The cloning method used was PERT (phenol emulsion reassociation technique), in which most male DNAs were absorbed by an excess amount of female DNAs to enrich Y-specific DNAs. Among 20 clones examined, one clone was male-specific. The others were gender-neutral; three of these were used as probes for internal controls. The male-specific clone was used as a probe to isolate other male-specific clones from a bovine male genomic DNA library. A clone thus obtained contained at least 3 male-specific EcoRI fragments. The original and the secondarily cloned DNAs were all sequenced. They were repetitive in nature. Oligonucleotides (20 mers) were synthesized on the basis of both the male-specific and gender-neutral DNA sequences as primers for PCR. A combination of 2 pairs of primers, i.e., male-specific and gender-neutral, gave discrete PCR products allowing discrimination between male and female DNA. The detection limit of the template DNA was 10 pg, equivalent to the DNA from 3 cells.
日本繁殖生物学会の論文