Format

Send to

Choose Destination
J Am Chem Soc. 2014 Nov 26;136(47):16582-7. doi: 10.1021/ja508527b. Epub 2014 Nov 14.

Nanopores discriminate among five C5-cytosine variants in DNA.

Author information

1
Department of Biomolecular Engineering, Baskin School of Engineering, MS SOE2, University of California , Santa Cruz, California 95064, United States.

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.

PMID:
25347819
PMCID:
PMC4277752
DOI:
10.1021/ja508527b
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for American Chemical Society Icon for PubMed Central
Loading ...
Support Center