Hiriyanna K T, Varkey J, Beer M, Benbow R M
Department of Zoology, Iowa State University of Science & Technology, Ames 50011-3223.
J Cell Biol. 1988 Jul;107(1):33-44. doi: 10.1083/jcb.107.1.33.
Biotinylated nucleotides (bio-11-dCTP, bio-11-dUTP, and bio-7-dATP) were microinjected into unfertilized and fertilized Xenopus laevis eggs. The amounts introduced were comparable to in vivo deoxy-nucleoside triphosphate pools. At various times after microinjection, DNA was extracted from eggs or embryos and subjected to electrophoresis on agarose gels. Newly synthesized biotinylated DNA was analyzed by Southern transfer and visualized using either the BluGENE or Detek-hrp streptavidin-based nucleic acid detection systems. Quantitation of the amount of biotinylated DNA observed at various times showed that the microinjected biotinylated nucleotides were efficiently incorporated in vivo, both into replicating endogenous chromosomal DNA and into replicating microinjected exogenous plasmid DNA. At least one biotinylated nucleotide could be incorporated in vivo for every eight nucleotides of DNA synthesized. Control experiments also showed that heavily biotinylated DNA was not subjected to detectable DNA repair during early embryogenesis (for at least 5 h after activation of the eggs). The incorporated biotinylated nucleotides were visualized by electron microscopy by using streptavidin-colloidal gold or streptavidin-ferritin conjugates to bind specifically to the biotin groups projecting from the newly replicated DNA. The incorporated biotinylated nucleotides were thus made visible as electron-dense spots on the underlying DNA molecules. Biotinylated nucleotides separated by 20-50 bases could be resolved. We conclude that nascent DNA synthesized in vivo in Xenopus laevis eggs can be visualized efficiently and specifically using the techniques described.
将生物素化核苷酸（生物素-11-dCTP、生物素-11-dUTP和生物素-7-dATP）显微注射到未受精和已受精的非洲爪蟾卵中。引入的量与体内脱氧核苷三磷酸池相当。显微注射后的不同时间，从卵或胚胎中提取DNA，并在琼脂糖凝胶上进行电泳。通过Southern转移分析新合成的生物素化DNA，并使用基于BluGENE或Detek-hrp链霉抗生物素蛋白的核酸检测系统进行可视化。对不同时间观察到的生物素化DNA量的定量分析表明，显微注射的生物素化核苷酸在体内能有效地掺入到复制的内源性染色体DNA和复制的显微注射外源性质粒DNA中。每合成八个DNA核苷酸，至少有一个生物素化核苷酸能在体内掺入。对照实验还表明，在早期胚胎发育过程中（卵激活后至少5小时），高度生物素化的DNA未受到可检测到的DNA修复。通过使用链霉抗生物素蛋白-胶体金或链霉抗生物素蛋白-铁蛋白偶联物与新复制DNA上突出的生物素基团特异性结合，通过电子显微镜观察掺入的生物素化核苷酸。因此，掺入的生物素化核苷酸在下面的DNA分子上呈现为电子致密斑点。相隔20 - 50个碱基的生物素化核苷酸可以分辨出来。我们得出结论，使用所述技术可以有效地、特异性地观察到非洲爪蟾卵体内合成的新生DNA。
