Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.
Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.
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DOI：
10.1371/journal.pone.0031597
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Marziali A
中科院分区：
文献类型：
--
作者：
Thompson JD;Shibahara G;Rajan S;Pel J;Marziali A
已知细胞群体中的罕见突变是许多疾病和癌症的标志。类似地，差异DNA甲基化模式出现在具有诊断潜力的罕见细胞群体中，例如在母体血液中循环的胎儿细胞。不幸的是，相对于野生型背景序列，具有诊断潜力的等位基因的频率通常远低于目前可用的序列分析方法（包括非常高通量的DNA测序）中的错误频率。我们证明了一种DNA制备和纯化方法，该方法通过在含有寡核苷酸探针的介质中进行非线性电泳分离，实现了具有单核苷酸特异性的靶DNA的10，000倍富集，以及通过单个胞嘧啶残基的甲基化而与背景不同的未修饰的甲基化DNA的100倍富集。
Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.