Weisenberger Daniel J, Trinh Binh N, Campan Mihaela, Sharma Shikhar, Long Tiffany I, Ananthnarayan Suchitra, Liang Gangning, Esteva Francisco J, Hortobagyi Gabriel N, McCormick Frank, Jones Peter A, Laird Peter W
Department of Surgery, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA 90033, USA.
Nucleic Acids Res. 2008 Aug;36(14):4689-98. doi: 10.1093/nar/gkn455. Epub 2008 Jul 15.
Alterations in cytosine-5 DNA methylation are frequently observed in most types of human cancer. Although assays utilizing PCR amplification of bisulfite-converted DNA are widely employed to analyze these DNA methylation alterations, they are generally limited in throughput capacity, detection sensitivity, and or resolution. Digital PCR, in which a DNA sample is analyzed in distributive fashion over multiple reaction chambers, allows for enumeration of discrete template DNA molecules, as well as sequestration of non-specific primer annealing templates into negative chambers, thereby increasing the signal-to-noise ratio in positive chambers. Here, we have applied digital PCR technology to bisulfite-converted DNA for single-molecule high-resolution DNA methylation analysis and for increased sensitivity DNA methylation detection. We developed digital bisulfite genomic DNA sequencing to efficiently determine single-basepair DNA methylation patterns on single-molecule DNA templates without an interim cloning step. We also developed digital MethyLight, which surpasses traditional MethyLight in detection sensitivity and quantitative accuracy for low quantities of DNA. Using digital MethyLight, we identified single-molecule, cancer-specific DNA hypermethylation events in the CpG islands of RUNX3, CLDN5 and FOXE1 present in plasma samples from breast cancer patients.
在大多数类型的人类癌症中，经常观察到5-胞嘧啶DNA甲基化的改变。尽管利用亚硫酸氢盐转化DNA的PCR扩增分析方法被广泛用于分析这些DNA甲基化改变，但它们通常在通量能力、检测灵敏度和/或分辨率方面受到限制。数字PCR以分布式方式在多个反应室中分析DNA样本，能够对离散的模板DNA分子进行计数，并将非特异性引物退火模板隔离到阴性反应室中，从而提高阳性反应室中的信噪比。在此，我们将数字PCR技术应用于亚硫酸氢盐转化的DNA，用于单分子高分辨率DNA甲基化分析以及提高DNA甲基化检测的灵敏度。我们开发了数字亚硫酸氢盐基因组DNA测序技术，无需中间克隆步骤即可有效确定单分子DNA模板上的单碱基对DNA甲基化模式。我们还开发了数字甲基化荧光定量PCR技术，其在检测灵敏度和低量DNA定量准确性方面超越了传统的甲基化荧光定量PCR技术。利用数字甲基化荧光定量PCR技术，我们在乳腺癌患者血浆样本中的RUNX3、CLDN5和FOXE1的CpG岛中鉴定出单分子、癌症特异性DNA高甲基化事件。
