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Determination of genomic 5-hydroxymethyl-2'-deoxycytidine in human DNA by capillary electrophoresis with laser induced fluorescence

Krais, Annette M LU ; Park, Yoon Jung; Plass, Christoph and Schmeiser, Heinz H. (2011) In Epigenetics 6(5). p.5-560
Abstract

Recent studies reported the presence of 5-hydroxymethylcytosine (5 hmC) as an additional modification in mammalian genomic DNA. To date, 5 hmC has been detected only in mouse DNA isolated from embryonic stem cells, some adult tissues and in DNA from human bone marrow. Understanding its biological function will require the development of sensitive analytical methods that allow the detection and quantification of 5-hydroxymethylcytosine along with 5-methylcytosine and cytosine. : Here we report the validation of a fast and sensitive method for the quantification of global 5-hydroxymethyl-2'-deoxycytidine (5 hmdC) in DNA. The method is based on a procedure consisting of fluorescence labeling of deoxyribonucleotides and analysis by... (More)

Recent studies reported the presence of 5-hydroxymethylcytosine (5 hmC) as an additional modification in mammalian genomic DNA. To date, 5 hmC has been detected only in mouse DNA isolated from embryonic stem cells, some adult tissues and in DNA from human bone marrow. Understanding its biological function will require the development of sensitive analytical methods that allow the detection and quantification of 5-hydroxymethylcytosine along with 5-methylcytosine and cytosine. : Here we report the validation of a fast and sensitive method for the quantification of global 5-hydroxymethyl-2'-deoxycytidine (5 hmdC) in DNA. The method is based on a procedure consisting of fluorescence labeling of deoxyribonucleotides and analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A double stranded DNA fragment containing a defined number of 5 hmdC residues was used for peak assignment, to establish separation conditions and to determine the limit of detection (LOD). The method yielded a LOD for 5 hmdC of 0.45 amol, which is equivalent to approximately to one 5 hmdC per 4,000 normal nucleotides (0.025%) using 1 μg of DNA as the matrix. : By applying the calibrated assay to the analysis of various DNAs we show that 5 hmdC is present in human tissue and human cancer cell lines. We demonstrate that by using CE-LIF DNA can be analyzed in one run for both methylation and hydroxymethylation of cytosine with high sensitivity and accuracy.

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author
publishing date
type
Contribution to journal
publication status
published
keywords
5-Methylcytosine, Cell Line, Tumor, DNA, DNA Methylation, Deoxycytidine, Electrophoresis, Capillary, Epigenesis, Genetic, Genome, Human, Humans, Lasers, Journal Article, Research Support, Non-U.S. Gov't, Validation Studies
in
Epigenetics
volume
6
issue
5
pages
6 pages
publisher
Landes Bioscience
external identifiers
  • scopus:79957579073
ISSN
1559-2294
DOI
10.4161/epi.6.5.15678
language
English
LU publication?
no
id
d7d8fcdf-fd8b-4da8-9d65-efa3f9f82b25
date added to LUP
2017-10-17 15:31:20
date last changed
2017-11-02 09:30:37
@article{d7d8fcdf-fd8b-4da8-9d65-efa3f9f82b25,
  abstract     = {<p>Recent studies reported the presence of 5-hydroxymethylcytosine (5 hmC) as an additional modification in mammalian genomic DNA. To date, 5 hmC has been detected only in mouse DNA isolated from embryonic stem cells, some adult tissues and in DNA from human bone marrow. Understanding its biological function will require the development of sensitive analytical methods that allow the detection and quantification of 5-hydroxymethylcytosine along with 5-methylcytosine and cytosine. : Here we report the validation of a fast and sensitive method for the quantification of global 5-hydroxymethyl-2'-deoxycytidine (5 hmdC) in DNA. The method is based on a procedure consisting of fluorescence labeling of deoxyribonucleotides and analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A double stranded DNA fragment containing a defined number of 5 hmdC residues was used for peak assignment, to establish separation conditions and to determine the limit of detection (LOD). The method yielded a LOD for 5 hmdC of 0.45 amol, which is equivalent to approximately to one 5 hmdC per 4,000 normal nucleotides (0.025%) using 1 μg of DNA as the matrix. : By applying the calibrated assay to the analysis of various DNAs we show that 5 hmdC is present in human tissue and human cancer cell lines. We demonstrate that by using CE-LIF DNA can be analyzed in one run for both methylation and hydroxymethylation of cytosine with high sensitivity and accuracy.</p>},
  author       = {Krais, Annette M and Park, Yoon Jung and Plass, Christoph and Schmeiser, Heinz H.},
  issn         = {1559-2294},
  keyword      = {5-Methylcytosine,Cell Line, Tumor,DNA,DNA Methylation,Deoxycytidine,Electrophoresis, Capillary,Epigenesis, Genetic,Genome, Human,Humans,Lasers,Journal Article,Research Support, Non-U.S. Gov't,Validation Studies},
  language     = {eng},
  number       = {5},
  pages        = {5--560},
  publisher    = {Landes Bioscience},
  series       = {Epigenetics},
  title        = {Determination of genomic 5-hydroxymethyl-2'-deoxycytidine in human DNA by capillary electrophoresis with laser induced fluorescence},
  url          = {http://dx.doi.org/10.4161/epi.6.5.15678},
  volume       = {6},
  year         = {2011},
}