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目的应用DNA芯片技术筛选人白血病K562细胞中的肿瘤特异性基因。方法提取正常人白细胞和K562细胞基因组DNA,并用限制性内切酶Sau3AI酶切。其中K562细胞基因组酶切产物经DNA聚合酶Ⅰ补平加A后克隆到T-载体,挑选阳性克隆,用PCR扩增,以纯化的PCR产物做探针,制备K562细胞基因组DNA芯片。正常人白细胞基因组酶切产物加上人工通用接头,用限制性标记技术标记上荧光标记物Cy3,与制备的K562细胞基因组DNA芯片杂交。结果芯片杂交结果经扫描分析,发现42个K562细胞肿瘤特异性基因,进一步用序列分析证实,其中有1个为BCR(breakpoint cluster gene)基因。结论我们自制的K562细胞基因组DNA芯片可以成功地用于筛选肿瘤特异性基因,为更好地从基因组水平研究肿瘤发生的分子机制提供了新的技术途径。
Objective To screen tumor-specific genes in human leukemia K562 cells by DNA microarray. Methods The genomic DNA of normal human leukocytes and K562 cells was extracted and digested with Sau3AI. K562 cells were digested with DNA polymerase I and then cloned into T-vector. The positive clones were selected and amplified by PCR. The purified PCR products were used as probes to prepare genomic DNA chips of K562 cells. The normal human leukocyte genome was digested with artificial universal linker, and the fluorescent marker Cy3 was labeled with a restriction-labeling technique to hybridize with the prepared K562 cell genomic DNA chip. Results The hybridization results of the chips were analyzed by scanning and found 42 tumor-specific genes of K562 cells were further confirmed by sequence analysis, of which 1 was a BCR (breakpoint cluster gene) gene. Conclusion Our home-made K562 cell genomic DNA microarray can be successfully used to screen tumor-specific genes and provide a new technical approach to better study the molecular mechanisms of tumorigenesis at the genome level.