title: A simple DNA extraction and rapid specific identification technique for single cells and early embryos of two breeds of Bos taurus.
abstract: In the process of nuclear transfer (NT), different cytoplasm from a donor cell and a recipient oocyte are mixed. However, it is unclear what effect the donor cytoplasm has upon the dedifferentiation of donor nuclei in enucleated ooplasm and upon subsequent production of live cloned offspring. Mitochondria are component parts of cytoplasm so the detection of mitochondrial DNA is helpful to reveal changes of donor cytoplasm in the NT reconstructed embryos. In this study, the experiments were designed to develop efficient DNA extraction techniques and specific primer pairs for mitochondrial DNA of Holstein and Chinese Yellow breeds in order to identify the changes of donor cytoplasm in early stage embryos. Firstly, by adding Triton X-100 and Taq DNA polymerase reaction buffer to the DNA extraction mixture, DNA was rapidly isolated from single diploid cells, single oocytes, early stage embryos and from single hairs. Secondly, two specific primer pairs for the two breeds were designed to detect the cytoplasmic DNA in a different amount of single cells and in early stage embryos. The results show that two specific fragments were successfully amplified from single somatic cells, single oocytes, parthenogenetic embryos and from NT reconstructed embryos. As a result, the techniques provide a powerful tool for studying the developmental mechanism in NT reconstructed embryos.
authors.affiliation: The Key Laboratory of Conservation Genetic and Reproductive Biology for Endangered Wild Animals of the Ministry of Education, Hangzhou 310029, PR China.
authors.forename: Qiu-Hong
authors.initials: QH
authors.lastname: Wan
authors.forename: Kai-Xian
authors.initials: KX
authors.lastname: Qian
authors.forename: Sheng-Guo
authors.initials: SG
authors.lastname: Fang
mesh_terms: D000818:Animals; D002417:Cattle / Q000196:embryology* / Q000235:genetics; D019976:Cloning, Organism; D003593:Cytoplasm / Q000502:physiology; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000032:analysis / Q000302:isolation & purification*; D004622:Embryo, Mammalian / Q000737:chemistry*; D008969:Molecular Sequence Data; D053652:Nuclear Transfer Techniques; D017830:Octoxynol; D009865:Oocytes / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D019914:Taq Polymerase
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial; D017830:Octoxynol; D019914:Taq Polymerase