DNA芯片技术筛选K562肿瘤细胞特异性基因

Application of microarrays in screening the tumor-specific genes in the genome of K562 cells

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 of K562 cells using DNA microarray technique. Methods The genomic DNA of normal white blood cells and cultured K562 cells were respectively purified and digested with Sau3AⅠ, and the digested DNA fragments of K562 cells were cloned into TA cloning vector to construct the corresponding genomic DNA library. The insert genomic DNA fragments were amplified from the library to prepare the microarray using Cartesian 5500 Microarrayer. The digested genomic DNA fragments of normal white blood cells were labeled with fluorescent Cy3 by restriction display PCR (RD-PCR), followed by hybridization with the microarray, after which the slide was washed and scanned with ScanArray. Results Among the 426 target genes, 42 differential genes were identified in the genomic DNA of K562 cells in comparison with the normal white blood cells. One of the genes was identified as the breakpoint cluster gene (BCR) after sequence analysis. Conclusions The DNA microarrays we constructed may effectively identify the tumor-specific genes in K562 cells, and DNA microarray technique can be helpful in elucidating the molecular mechanisms of tumorigenesis at the genomic level.