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Nanopores discriminate among five C5-cytosine variants in DNA.

Authors :
Wescoe ZL
Schreiber J
Akeson M
Source :
Journal of the American Chemical Society [J Am Chem Soc] 2014 Nov 26; Vol. 136 (47), pp. 16582-7. Date of Electronic Publication: 2014 Nov 14.
Publication Year :
2014

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 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.

Details

Language :
English
ISSN :
1520-5126
Volume :
136
Issue :
47
Database :
MEDLINE
Journal :
Journal of the American Chemical Society
Publication Type :
Academic Journal
Accession number :
25347819
Full Text :
https://doi.org/10.1021/ja508527b