COLD-PCR amplification of bisulfite-converted DNA allows the enrichment and sequencing of rare un-methylated genomic regions.
COLD-PCR amplification of bisulfite-converted DNA allows the enrichment and sequencing of rare un-methylated genomic regions.
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DOI：
10.1371/journal.pone.0094103
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Merewood A
中科院分区：
文献类型：
--
作者：
Castellanos-Rizaldos E;Milbury CA;Karatza E;Chen CC;Makrigiorgos GM;Merewood A
DNA的异常低甲基化在包括癌症和糖尿病在内的一系列人类疾病中都很明显。开发能够检测甲基化样本中微量未甲基化DNA的灵敏分析方法在几种情况下都是有用的。在这里，我们描述了一种新的方法，快速冷-MS-PCR，它优先扩增未甲基化的DNA序列。通过在双亚硫酸盐转化的DNA的PCR过程中采用适当的变性温度，FAST-COLD-MS-PCR可以丰富未甲基化的DNA，并使差示熔融分析或亚硫酸氢盐测序成为可能。以MGMT基因启动子上的甲基化为模型，研究表明，受控甲基化样本的连续稀释导致对未甲基化序列的可靠测序，低至0.05%的未甲基化到甲基化的DNA。对临床胶质瘤和婴儿血液样本的筛选表明，非甲基化DNA对甲基化DNA的浓缩程度可以通过选择变性温度来调节，这为分析部分甲基化DNA或揭示未甲基化DNA的痕迹提供了一种方便的方法。FAST-COLD-MS-PCR可用于检测癌症、糖尿病或饮食相关甲基化改变中的甲基化缺失/印记。
Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes.
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影响因子：
3.9
作者：
Li, Jin;Milbury, Coren A.;Li, Cheng;Makrigiorgos, G. Mike
通讯作者：
Makrigiorgos, G. Mike
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：
9.3
作者：
Bonanno, Cinzia;Shehi, Erlet;Makrigiorgos, G. Mike
通讯作者：
Makrigiorgos, G. Mike
影响因子：
4.5
作者：
McGraw S;Oakes CC;Martel J;Cirio MC;de Zeeuw P;Mak W;Plass C;Bartolomei MS;Chaillet JR;Trasler JM
通讯作者：
Trasler JM
影响因子：
4.8
作者：
Fisher, Marisa M.;Chumbiauca, Cristina N. Perez;Tersey, Sarah A.
通讯作者：
Tersey, Sarah A.