A Modified and Improved Method for Bisulphite Based Cytosine Methylation Analysis

作者：Alexander Olek, Joachim Oswald, Jörn Walter · 发表于：Nucleic Acids Research · 年份：1996 · DOI：10.1093/nar/24.24.5064 · 被引用次数：375 · 研究领域：Epigenetics and DNA Methylation、RNA modifications and cancer、Genetic Syndromes and Imprinting

Sequencing of bisulphite modified genomic DNA is the most powerful method to determine methylation patterns in chromosomal DNA. In many experimental systems, the amount of material available for analysis is very small which makes it necessary to perform experiments at extreme levels of sensitivity and reproducibility. In this communication, we present an improved modification of the bisulphite based sequencing method. Our strategy is to perform the bisulphite treatment and subsequent PCR steps on material embedded into agarose beads. This prevents loss of DNA during the experimental procedure and ensures an optimal bisulphite reactivity by maintaining the DNA in the single stranded form. The modification improves previously published protocols in that it facilitates the handling of probes and reproducibly reaches a very high level of sensitivity.