| 1. |
- Douvlataniotis, Karolos, et al.
(författare)
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No evidence for DNA N-6-methyladenine in mammals
- 2020
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Ingår i: Science Advances. - Washington, DC, United States : American Association for the Advancement of Science (A A A S). - 2375-2548. ; 6:12
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Tidskriftsartikel (refereegranskat)abstract
- N-6-methyladenine (6mdA) is a widespread DNA modification in bacteria. More recently, 6mdA has also been characterized in mammalian DNA. However, measurements of 6mdA abundance and profiles are often very dissimilar between studies, even when performed on DNA from identical mammalian cell types. Using comprehensive bioinformatics analyses of published data and novel experimental approaches, we reveal that efforts to assay 6mdA in mammals have been severely compromised by bacterial contamination, RNA contamination, technological limitations, and antibody nonspecificity. These complications render 6mdA an exceptionally problematic DNA modification to study and have resulted in erroneous detection of 6mdA in several mammalian systems. Together, our results strongly imply that the evidence published to date is not sufficient to support the presence of 6mdA in mammals.
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| 2. |
- Lentini, Antonio, et al.
(författare)
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A reassessment of DNA-immunoprecipitation-based genomic profiling
- 2018
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Ingår i: Nature Methods. - : NATURE PUBLISHING GROUP. - 1548-7091 .- 1548-7105. ; 15:7, s. 499-
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Tidskriftsartikel (refereegranskat)abstract
- DNA immunoprecipitation followed by sequencing (DIP-seq) is a common enrichment method for profiling DNA modifications in mammalian genomes. However, the results of independent DIP-seq studies often show considerable variation between profiles of the same genome and between profiles obtained by alternative methods. Here we show that these differences are primarily due to the intrinsic affinity of IgG for short unmodified DNA repeats. This pervasive experimental error accounts for 50-99% of regions identified as enriched for DNA modifications in DIP-seq data. Correction of this error profoundly altered DNA-modification profiles for numerous cell types, including mouse embryonic stem cells, and subsequently revealed novel associations among DNA modifications, chromatin modifications and biological processes. We conclude that both matched input and IgG controls are essential in order for the results of DIP-based assays to be interpreted correctly, and that complementary, non-antibody-based techniques should be used to validate DIP-based findings to avoid further misinterpretation of genome-wide profiling data.
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