Fetni R, Drouin R, Lemieux N, Messier P E, Richer C L
Département d'Anatomie, Université de Montréal, PQ, Canada.
Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10916-20. doi: 10.1073/pnas.88.23.10916.
Electron microscopy (EM) is seldom used with in situ hybridization to localize DNA sequences because banding methods for chromosome identification could not be coupled to EM techniques. We have applied an immunochemical replication-banding method specific for EM to solve this problem. A thymidine synchronization/BrdUrd release protocol allows BrdUrd incorporation only into late replicating bands. A biotinylated DNA probe is hybridized in situ to its complementary sequence. The biotinylated probe and the BrdUrd-substituted DNA are simultaneously localized by different reporter/detection systems using different-sized colloidal gold particles as electron-dense tags. We demonstrate the high precision of this mapping procedure by localizing on long prophase chromosomes (greater than 1000 bands per haploid set) the pXBR-1 sequence to a small subregion of the centromeric subband Xp11.1-Xq11.1. This localization to a part of an individual prophase subband is the most precise localization ever reported on human banded mitotic chromosomes.
电子显微镜（EM）很少与原位杂交联用定位DNA序列，因为用于染色体识别的显带方法无法与EM技术相结合。我们应用了一种专门用于EM的免疫化学复制显带方法来解决这一问题。胸腺嘧啶同步/5-溴脱氧尿苷（BrdUrd）释放方案可使BrdUrd仅掺入晚复制带。生物素化的DNA探针原位杂交至其互补序列。生物素化探针和BrdUrd取代的DNA通过不同的报告/检测系统，使用不同大小的胶体金颗粒作为电子致密标记物同时进行定位。我们通过将pXBR-1序列定位到中期染色体（单倍体组每条染色体大于1000条带）着丝粒亚带Xp11.1-Xq11.1的一个小亚区域，证明了这种定位程序的高精度。这种定位到单个早中期亚带的一部分是人类有丝分裂显带染色体上报道过的最精确的定位。
