State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
Clin Chim Acta. 2010 Sep 6;411(17-18):1187-94. doi: 10.1016/j.cca.2010.03.026. Epub 2010 Mar 27.
Microarray technology combining with bisulfite-PCR offers a high-throughput approach for detection of DNA methylation. However, the use of microarray-based DNA methylation analysis has been limited by the low throughput of sample preparation due to the difficulty in simultaneous amplification of multiple targets.
A set of target-selection-padlock probes was designed to capture the target sequences containing the queried CpG sites from bisulfite-treated genomic DNA. Then all targets were simultaneously amplified by a pair of common primers. The methylation status of multiple targets was detected by single base extension (SBE) on oligonucleotide microarray based on polyacrylic acid-covered surface.
This assay has been successfully applied to analyze promoter methylation of 8 tumor suppressor genes in 12 colorectal cancer samples and 2 normal control samples. The target-selection-padlock probe exhibited both high specificity and high efficiency for the parallel amplification of multiple genes. The accurate and high-throughput detection for DNA methylation was achieved by a combination of target-selection-padlock probes and microarray.
The present study provides a robust and accurate assay for DNA methylation status of multiple genes. This method may be useful for a large-scale screen of DNA methylation in cancer cell lines and clinical samples.
微阵列技术与亚硫酸氢盐-PCR 相结合，为检测 DNA 甲基化提供了一种高通量的方法。然而，由于难以同时扩增多个靶标，基于微阵列的 DNA 甲基化分析的应用受到了样品制备通量低的限制。
设计了一组靶标选择发夹探针，用于从经亚硫酸氢盐处理的基因组 DNA 中捕获包含查询 CpG 位点的靶序列。然后，通过一对通用引物同时对所有靶标进行扩增。通过基于聚丙烯酸覆盖表面的寡核苷酸微阵列上的单碱基延伸（SBE）检测多个靶标的甲基化状态。
该测定法已成功应用于分析 12 例结直肠癌样本和 2 例正常对照样本中 8 个肿瘤抑制基因的启动子甲基化。靶标选择发夹探针在多个基因的平行扩增中表现出高特异性和高效率。通过靶标选择发夹探针和微阵列的组合实现了 DNA 甲基化的准确和高通量检测。
本研究提供了一种用于检测多个基因 DNA 甲基化状态的稳健而准确的方法。该方法可用于癌症细胞系和临床样本中大规模的 DNA 甲基化筛选。
