Weier H U, Kleine H D, Gray J W
Biomedical Sciences Division, University of California, Lawrence Livermore National Laboratory 94550.
Hum Genet. 1991 Aug;87(4):489-94. doi: 10.1007/BF00197174.
Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.
合成了优先与人8号染色体着丝粒区域结合的探针DNA。通过使用聚合酶链反应（PCR）的体外DNA扩增，从分选纯化的人8号染色体中选择性扩增α卫星探针DNA分子。在PCR过程中通过掺入生物素化的脱氧尿苷进行探针标记。未纯化的由α卫星单体和更高分子量DNA片段组成的探针DNA与中期染色体铺展进行原位杂交，结果显示其与许多染色体的着丝粒区域结合。然而，用未标记的总人α卫星DNA进行封闭可显著减少与8号以外染色体的交叉杂交。在这些条件下，简并探针DNA使得在中期铺展和间期细胞核中能够清晰地观察到8号染色体着丝粒DNA所占据的区域，从而极大地促进了肿瘤细胞中染色体数目畸变的检测。对大小分级的α卫星DNA进行原位杂交确定单体部分是交叉杂交的主要原因。α卫星二聚体和更高分子量DNA片段对人8号染色体显示出相对较高的特异性。
