Bicknell D C, Markie D, Spurr N K, Bodmer W F
Cancer Immunology Laboratory, University of Oxford, John Radcliffe Hospital, Headington, United Kingdom.
Genomics. 1991 May;10(1):186-92. doi: 10.1016/0888-7543(91)90499-5.
The ubiquitous nature of the Alu sequence throughout the human genome forms the basis of an assay we present here for analyzing the human chromosome content of human x rodent somatic cell hybrids. A human-specific Alu primer was used both to amplify sequences and to 32P label the products in a polymerase chain reaction (PCR) technique. Unlabeled inter-Alu PCR products from two series of human x rodent hybrids were used to prepare dot blots which were probed with labeled inter-Alu products prepared from between 10(3) and 10(4) hybrid cells. In the first series we demonstrate that a labeled inter-Alu probe from the hybrid DL18ts, containing a single chromosome 18, on a dot blot hybridized only with those inter-Alu products containing chromosome 18. Similar specificity for human chromosome 5 was shown when a Southern blot of the PCR products was hybridized with a probe made from the hybrid HHW 213, which contains only chromosome 5p. Using a dot blot from a second series of control hybrids, 15 of which contained single human chromosomes, hybridization of a labeled probe from the hybrid 18X4-1 was shown to react specifically with the controls that expressed chromosome 18. Application of the technique reported here allows simple and rapid characterization of the human chromosome content in human x rodent hybrids.
Alu序列在整个人类基因组中普遍存在,这构成了我们在此介绍的一种分析人-啮齿动物体细胞杂种中人染色体组成的检测方法的基础。在聚合酶链反应(PCR)技术中,使用了一种人类特异性Alu引物来扩增序列并对产物进行32P标记。来自两系列人-啮齿动物杂种的未标记的Alu间PCR产物用于制备斑点印迹,并用由10(3)至10(4)个杂种细胞制备的标记Alu间产物进行杂交检测。在第一系列实验中,我们证明,在斑点印迹上,来自含有单条18号染色体的杂种DL18ts的标记Alu间探针仅与那些含有18号染色体的Alu间产物杂交。当PCR产物的Southern印迹与由仅含有5号染色体短臂的杂种HHW 213制备的探针杂交时,显示出对5号人类染色体有类似的特异性。使用来自第二系列对照杂种(其中15个含有单条人类染色体)的斑点印迹,显示来自杂种18X4-1的标记探针的杂交与表达18号染色体的对照特异性反应。本文报道的技术应用能够简单快速地鉴定人-啮齿动物杂种中的人染色体组成。