Recovery of bisulfite-converted genomic sequences in the methylation-sensitive QPCR.
Recovery of bisulfite-converted genomic sequences in the methylation-sensitive QPCR.
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DOI：
10.1093/nar/gkm055
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发表时间：
2007
影响因子：
14.9
通讯作者：
Smith SS
中科院分区：
文献类型：
--
作者：
Munson K;Clark J;Lamparska-Kupsik K;Smith SS
目前已经出现了多种检测基因组DNA甲基化状态的方法。目前，几乎所有这类方法都使用亚硫酸氢盐介导的变性DNA脱氨基。虽然这种治疗有效地将胞嘧啶脱氨基为尿嘧啶，使大多数5-甲基胞嘧啶保持不变，但它也引入了产生大量DNA单链断裂的基本位点。我们研究了这两个过程的相互作用，以确定它们对甲基化敏感的QPCR方法的相对影响。输入DNA的切割程度是显著的，并且似乎是DNA浓度的递增函数。即便如此，研究结果表明，62-nT靶标中只有10%的∼会因降解而丢失，而131nT以下的靶标只会损失20%。在随后的亚硫酸盐去除和脱磺步骤中发现发生了更显著的损失，这似乎是在最常用的方法中定量聚合酶链式反应之前所需的与基质结合和洗脱相关的尺寸选择性的结果。对于产生1 μg脱氧核糖核酸的生物样本，这些发现表明，在目前的MS-QPCR实施中，亚硫酸氢盐处理会导致低回收率，从而排除了对DNA甲基化模式的可靠分析，而无论目标大小如何。
Many methods for the detection of genomic DNA methylation states have appeared. Currently, nearly all such methods employ bisulfite-mediated deamination of denatured DNA. While this treatment effectively deaminates cytosines to uracils, leaving most 5-methylcytosines intact, it also introduces abasic sites that generate a significant number of single-strand breaks in DNA. We have investigated the interplay of these two processes in order to determine their relative effects on the methylation-sensitive QPCR method. The extent of cleavage of the input DNA is significant and appears to be an increasing function of DNA concentration. Even so, the results suggest that only ∼10% of a 62-nt target will be lost due to degradation and targets up to 131 nt will suffer only a 20% loss. More significant losses were found to occur during the subsequent removal of bisulfite and desulfonation steps that appear to be the result of size selectivity associated with matrix binding and elution required prior to QPCR in the most commonly used protocols. For biospecimens yielding <1 μg of DNA, these findings suggest that bisulfite treatment, in current implementations of MS-QPCR, result in low recoveries that preclude reliable analysis of DNA methylation patterns regardless of target size.
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DOI：
10.1073/pnas.93.18.9821
发表时间：
1996-09-03
影响因子：
11.1
作者：
Herman, JG;Graff, JR;Baylin, SB
通讯作者：
Baylin, SB
影响因子：
14.9
作者：
Gonzalgo, ML;Jones, PA
通讯作者：
Jones, PA
影响因子：
14.9
作者：
Olek, A;Oswald, J;Walter, J
通讯作者：
Walter, J
影响因子：
2.9
作者：
Chen, ZX;Riggs, AD
通讯作者：
Riggs, AD
影响因子：
5.8
作者：
Hoque, MO;Rosenbaum, E;Umbricht, CB
通讯作者：
Umbricht, CB