Nanopores discriminate among five C5-cytosine variants in DNA.
Nanopores discriminate among five C5-cytosine variants in DNA.
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DOI：
10.1021/ja508527b
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发表时间：
2014-11-26
影响因子：
15
通讯作者：
Akeson, Mark
中科院分区：
文献类型：
--
作者：
Wescoe, Zachary L.;Schreiber, Jacob;Akeson, Mark
单个DNA分子可以使用连接到过程酶的纳米孔以单核苷酸的精度进行读取。已经实现了对四个典型碱基的区分，以及对胞嘧啶、5-甲基胞嘧啶(MC)和5-羟甲基胞嘧啶(HMC)的区分。在哺乳动物细胞中，HMC在酶促转化为胞嘧啶的过程中还会产生另外两个修饰的胞嘧啶碱基，即5-羧基胞嘧啶(CAC)和5-甲酰胞嘧啶(FC)。因此，对靶细胞胞嘧啶表观遗传状态的准确描述也应该包括这些C5胞嘧啶变异体。在本研究中，我们使用膜片钳放大器来获取由phi29 DNA聚合酶控制的DNA模板通过M2MspA孔的移位引起的离子电流示踪。基于三个连续离子流态的决策边界被用于调用∼4400DNA分子中Cg二核苷酸上的MC、HMC、CAC、FC或胞嘧啶。我们发现，单遍读取的正确基本调用百分比在91.6%到98.3%之间。这种准确性依赖于CG二核苷酸周围最近邻碱基的同一性。
Individual DNA molecules can be read at single nucleotide precision using nanopores coupled to processive enzymes. Discrimination among the four canonical bases has been achieved, as has discrimination among cytosine, 5-methylcytosine (mC), and 5-hydroxymethylcytosine (hmC). Two additional modified cytosine bases, 5-carboxylcytosine (caC) and 5-formylcytosine (fC), are produced during enzymatic conversion of hmC to cytosine in mammalian cells. Thus, an accurate picture of the cytosine epigenetic status in target cells should also include these C5-cytosine variants. In the present study, we used a patch clamp amplifier to acquire ionic current traces caused by phi29 DNA polymerase-controlled translocation of DNA templates through the M2MspA pore. Decision boundaries based on three consecutive ionic current states were implemented to call mC, hmC, caC, fC, or cytosine at CG dinucleotides in ∼4400 individual DNA molecules. We found that the percentage of correct base calls for single pass reads ranged from 91.6% to 98.3%. This accuracy depended upon the identity of nearest neighbor bases surrounding the CG dinucleotide.
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影响因子：
48
作者：
Flusberg, Benjamin A.;Webster, Dale R.;Lee, Jessica H.;Travers, Kevin J.;Olivares, Eric C.;Clark, Tyson A.;Korlach, Jonas;Turner, Stephen W.
通讯作者：
Turner, Stephen W.
DOI：
10.1073/pnas.1310615110
发表时间：
2013-11-19
影响因子：
11.1
作者：
Schreiber, Jacob;Wescoe, Zachary L.;Akeson, Mark
通讯作者：
Akeson, Mark
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
21.8
作者：
通讯作者：
--
影响因子：
7.5
作者：
Geurts, P;Ernst, D;Wehenkel, L
通讯作者：
Wehenkel, L