Olek A, Oswald J, Walter J
Max-Planck-Institut für Molekulare Genetik, Berlin, Germany.
Nucleic Acids Res. 1996 Dec 15;24(24):5064-6. doi: 10.1093/nar/24.24.5064.
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.
亚硫酸氢盐修饰的基因组DNA测序是确定染色体DNA甲基化模式的最有效方法。在许多实验系统中，可用于分析的材料量非常少，这使得有必要在极高的灵敏度和可重复性水平上进行实验。在本通讯中，我们提出了一种基于亚硫酸氢盐测序方法的改进方法。我们的策略是对嵌入琼脂糖珠的材料进行亚硫酸氢盐处理和随后的PCR步骤。这可防止DNA在实验过程中丢失，并通过将DNA保持在单链形式来确保最佳的亚硫酸氢盐反应性。该改进方法改进了先前发表的方案，因为它便于处理探针，并可重复地达到非常高的灵敏度水平。
