Liang Gangning, Gonzalgo Mark L, Salem Carol, Jones Peter A
Department of Biochemistry and Molecular Biology, Urologic Cancer Research Laboratory, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA.
Methods. 2002 Jun;27(2):150-5. doi: 10.1016/s1046-2023(02)00068-3.
The ability to detect methylation changes associated with oncogenic transformation is of critical importance in understanding how DNA methylation may contribute to tumorigenesis. We have developed a simple and reproducible fingerprinting method called methylation-sensitive arbitrarily primed polymerase chain reaction (AP-PCR) to screen for DNA methylation changes. This technique relies on digesting genomic DNA with methylation-sensitive and -insensitive restriction enzymes (e.g., HpaII and MspI) prior to AP-PCR amplification. Matched normal and tumor DNAs were compared to identify differential methylation. After the PCR products were resolved on high-resolution polyacrylamide gels, regions of genomic DNA that showed hypo- and hypermethylation associated with tumors were detected. These fragments were then isolated, cloned, and sequenced. Novel CpG islands were found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.
检测与致癌转化相关的甲基化变化的能力对于理解DNA甲基化如何促进肿瘤发生至关重要。我们开发了一种简单且可重复的指纹图谱方法，称为甲基化敏感任意引物聚合酶链反应（AP-PCR），用于筛选DNA甲基化变化。该技术依赖于在AP-PCR扩增之前用甲基化敏感和不敏感的限制性内切酶（如HpaII和MspI）消化基因组DNA。比较匹配的正常和肿瘤DNA以鉴定差异甲基化。在将PCR产物在高分辨率聚丙烯酰胺凝胶上分离后，检测到与肿瘤相关的基因组DNA的低甲基化和高甲基化区域。然后分离、克隆和测序这些片段。发现新的CpG岛在膀胱和结肠肿瘤中经常发生高甲基化。我们已经证明，该技术是一种快速有效的方法，可用于筛选基因组DNA中改变的甲基化模式，并分离与这些变化相关的特定序列。
