Umetani Naoyuki, de Maat Michiel F G, Mori Takuji, Takeuchi Hiroya, Hoon Dave S B
Department of Molecular Oncology, Martin H. Well Laboratory, John Wayne Cancer Institute, Santa Monica, CA 90404, USA.
Biochem Biophys Res Commun. 2005 Apr 1;329(1):219-23. doi: 10.1016/j.bbrc.2005.01.088.
Optimization of highly sensitive methods to detect methylation of CpG islands in gene promoter regions requires adequate methylated and unmethylated control DNA. Whereas universal methylated control DNA is available, universal unmethylated control (UUC) DNA has not been made because demethylase is not available to remove methyl groups from all methylated cytosines. On the basis that DNA synthesized by DNA polymerase does not contain methylated cytosines, we developed a method to create UUC DNA by nested whole genome amplification (WGA) with phi29 DNA polymerase. Contamination of the template genomic DNA in UUC was only 3.1 x 10(-7), below the detection limit of sensitive methods used for methylation studies such as methylation-specific PCR. Assessment of microsatellite markers demonstrated that even nested phi29 WGA achieves highly accurate and homogeneous amplification with very low amounts of genomic DNA as an initial template. The UUC DNA created by nested phi29 WGA is practically very useful for methylation analysis.
优化用于检测基因启动子区域CpG岛甲基化的高灵敏度方法需要合适的甲基化和未甲基化对照DNA。虽然通用甲基化对照DNA已有，但通用未甲基化对照（UUC）DNA尚未制备，因为没有脱甲基酶可从所有甲基化胞嘧啶上去除甲基基团。基于DNA聚合酶合成的DNA不包含甲基化胞嘧啶这一事实，我们开发了一种通过使用phi29 DNA聚合酶进行巢式全基因组扩增（WGA）来创建UUC DNA的方法。UUC中模板基因组DNA的污染仅为3.1×10（-7），低于用于甲基化研究的灵敏方法（如甲基化特异性PCR）的检测限。微卫星标记评估表明，即使是巢式phi29 WGA，以极少量的基因组DNA作为初始模板也能实现高度准确和均匀的扩增。通过巢式phi29 WGA创建的UUC DNA在甲基化分析中非常实用。
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