Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies.
Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies.
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DNA 亚硫酸氢盐转化中的错误：调节不适当和失败的转化频率。
DOI：
10.1093/nar/gkn691
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发表时间：
2008-12
影响因子：
14.9
通讯作者：
Laird CD
中科院分区：
文献类型：
--
作者：
Genereux DP;Johnson WC;Burden AF;Stöger R;Laird CD
亚硫酸氢盐处理可用于确定DNA中单个胞嘧啶的甲基化状态。理想情况下，亚硫酸氢盐处理将未甲基化的胞嘧啶脱氨基为尿嘧啶，并使5-甲基胞嘧啶保持不变。发生两种类型的亚硫酸氢盐转化错误：5-甲基胞嘧啶转化为胸腺嘧啶的不适当转化，以及未甲基化胞嘧啶转化为尿嘧啶的失败。传统的亚硫酸氢盐处理需要数小时暴露于低摩尔浓度、低温亚硫酸氢盐（“LowMT”），有时还需要热变性。据报道，另一种高摩尔浓度高温（“HighMT”）方案可加速转化并减少不适当的转化。我们使用分子编码来获得关于单链和发夹连接寡核苷酸的LowMT和HighMT治疗的失败和不适当转换频率的经验证的个体分子数据。在考虑了与亚硫酸氢盐无关的误差之后，我们发现：（i）在暴露于亚硫酸氢盐的分子已经达到完全或接近完全转化之后，不适当的转化事件主要在暴露于亚硫酸氢盐的分子上累积;（ii）HighMT处理是优选的，因为它在转化率中在位点之间和分子之间产生更大的均一性，从而产生更可靠的数据;（iii）亚硫酸氢盐处理的不同持续时间将产生适合于解决不同实验问题的数据;和（iv）转换误差可用于评估在没有分子编码的益处的情况下收集的甲基化数据的有效性。
Bisulfite treatment can be used to ascertain the methylation states of individual cytosines in DNA. Ideally, bisulfite treatment deaminates unmethylated cytosines to uracils, and leaves 5-methylcytosines unchanged. Two types of bisulfite-conversion error occur: inappropriate conversion of 5-methylcytosine to thymine, and failure to convert unmethylated cytosine to uracil. Conventional bisulfite treatment requires hours of exposure to low-molarity, low-temperature bisulfite (‘LowMT’) and, sometimes, thermal denaturation. An alternate, high-molarity, high-temperature (‘HighMT’) protocol has been reported to accelerate conversion and to reduce inappropriate conversion. We used molecular encoding to obtain validated, individual-molecule data on failed- and inappropriate-conversion frequencies for LowMT and HighMT treatments of both single-stranded and hairpin-linked oligonucleotides. After accounting for bisulfite-independent error, we found that: (i) inappropriate-conversion events accrue predominantly on molecules exposed to bisulfite after they have attained complete or near-complete conversion; (ii) the HighMT treatment is preferable because it yields greater homogeneity among sites and among molecules in conversion rates, and thus yields more reliable data; (iii) different durations of bisulfite treatment will yield data appropriate to address different experimental questions; and (iv) conversion errors can be used to assess the validity of methylation data collected without the benefit of molecular encoding.
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影响因子：
56.9
作者：
Jacobsen, SE;Meyerowitz, EM
通讯作者：
Meyerowitz, EM
DOI：
10.1002/ssu.2980030304
发表时间：
1987-01-01
期刊：
SEMINARS IN SURGICAL ONCOLOGY
影响因子：
--
作者：
FEINBERG, AP;VOGELSTEIN, B
通讯作者：
VOGELSTEIN, B
影响因子：
64.5
作者：
SWAIN, JL;STEWART, TA;LEDER, P
通讯作者：
LEDER, P
DOI：
10.1073/pnas.0604602103
发表时间：
2006-09-19
影响因子：
11.1
作者：
Egger, Gerda;Jeong, Shinwu;Liang, Gangning
通讯作者：
Liang, Gangning
影响因子：
30.8
作者：
CLARK, SJ;HARRISON, J;FROMMER, M
通讯作者：
FROMMER, M