Abstract
In order to establish a strategy to construct a DNA library from a specific region of a chromosome, bovine Y-chromosome DNA was amplified by the microdissection of whole chromosomes and polymerase chain reaction (PCR). Intact Y-chromosomes were microdissected from metaphase lymphocyte chromosomes and were digested with Proteinase K, followed by DNA extraction with phenol/chloroform. Purified DNA was cleaved with the restriction enzyme Sau3AI and the fragments were ligated to a synthetic linker-primer into which EcoRI site composite was introduced. The resultant Y-chromosome nucleotide sequences were amplified by PCR and cloned with vector DNA (EcoRI-digested pUC19 DNA). Sau3AI-digested pUC19 DNA served as a positive control and could be amplified by PCR using at least 0.2 pg of DNA. The Y-chromosome was successfully microdissected under an inverted microscope (400 ×), DNA was amplified by the PCR method and a smear of DNA (0.15-1.0 kb) was obtained. Two of sixty-five clones (0.05-1.2 kb) were male-specific by dot hybridization to male and female genomic DNA but no clone was found to be Y-chromosome specific by Southern hybridization.
