Title |
Nanopores Discriminate among Five C5-Cytosine Variants in DNA
|
---|---|
Published in |
Journal of the American Chemical Society, November 2014
|
DOI | 10.1021/ja508527b |
Pubmed ID | |
Authors |
Zachary L. Wescoe, Jacob Schreiber, Mark Akeson |
Abstract |
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 cytosine epigenetic status in target cells must 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 ~ 4,400 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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United Kingdom | 3 | 15% |
France | 1 | 5% |
Australia | 1 | 5% |
Switzerland | 1 | 5% |
Unknown | 9 | 45% |
Demographic breakdown
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Scientists | 8 | 40% |
Practitioners (doctors, other healthcare professionals) | 1 | 5% |
Science communicators (journalists, bloggers, editors) | 1 | 5% |
Mendeley readers
Geographical breakdown
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Korea, Republic of | 1 | <1% |
India | 1 | <1% |
Belgium | 1 | <1% |
United Kingdom | 1 | <1% |
Japan | 1 | <1% |
Russia | 1 | <1% |
Unknown | 199 | 96% |
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Readers by professional status | Count | As % |
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Student > Ph. D. Student | 51 | 25% |
Researcher | 30 | 14% |
Student > Master | 28 | 14% |
Student > Bachelor | 24 | 12% |
Professor > Associate Professor | 11 | 5% |
Other | 29 | 14% |
Unknown | 34 | 16% |
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Agricultural and Biological Sciences | 35 | 17% |
Chemistry | 26 | 13% |
Engineering | 15 | 7% |
Physics and Astronomy | 12 | 6% |
Other | 26 | 13% |
Unknown | 36 | 17% |