Mill Jonathan, Petronis Arturas
The Krembil Family Epigenetics Laboratory, Centre for Addiction and Mental Health, Toronto, Ontario, Canada.
Methods Mol Biol. 2009;507:371-81. doi: 10.1007/978-1-59745-522-0_27.
noindent Sodium bisulfite modification-based fine mapping of methylated cytosines represents the gold standard technique for DNA methylation studies. A major problem with this approach, however, is that it results in considerable DNA degradation, and large quantities of genomic DNA material are needed if numerous genomic regions are to be profiled. This chapter describes a method for profiling DNA methylation from small amounts of genomic-DNA starting material utilizing an efficient sodium bisulfite conversion method followed by whole-genome amplification (WGA). WGA is a useful method to overcome the problem of low initial amount of DNA and/or severe DNA degradation during conventional sodium bisulfite treatment in studies investigating DNA methylation. WGA is a relatively inexpensive process that can be optimized for high-throughput application and enables the thorough investigation of methylation at numerous genomic locations on samples for which DNA availability is low. Data from our lab has demonstrated that bisulfite-treated DNA amplified using WGA can be used for a range of downstream DNA methylation mapping procedures, including bisulfite-primer optimization, the sequencing of cloned PCR products, MS-SNuPE, and Pyrosequencing.
基于亚硫酸氢钠修饰的甲基化胞嘧啶精细定位是DNA甲基化研究的金标准技术。然而，这种方法的一个主要问题是它会导致相当程度的DNA降解，如果要对大量基因组区域进行分析，就需要大量的基因组DNA材料。本章介绍了一种从少量基因组DNA起始材料分析DNA甲基化的方法，该方法利用高效的亚硫酸氢钠转化方法，随后进行全基因组扩增（WGA）。在研究DNA甲基化时，WGA是一种有用的方法，可克服传统亚硫酸氢钠处理过程中DNA初始量低和/或严重DNA降解的问题。WGA是一个相对便宜的过程，可以针对高通量应用进行优化，并且能够对DNA可用性低的样本在众多基因组位置的甲基化进行全面研究。我们实验室的数据表明，使用WGA扩增的亚硫酸氢钠处理后的DNA可用于一系列下游DNA甲基化定位程序，包括亚硫酸氢钠引物优化、克隆PCR产物测序、MS-SNuPE和焦磷酸测序。
