Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation.
Identifieur interne : 001329 ( Main/Curation ); précédent : 001328; suivant : 001330Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation.
Auteurs : Tyson A. Clark [États-Unis] ; Xingyu Lu ; Khai Luong ; Qing Dai ; Matthew Boitano ; Stephen W. Turner ; Chuan He ; Jonas KorlachSource :
- BMC biology [ 1741-7007 ] ; 2013.
Descripteurs français
- KwdFr :
- 5-Méthyl-cytosine (métabolisme), Analyse de séquence d'ADN (MeSH), Cinétique (MeSH), DNA modification methylases (métabolisme), Escherichia coli (enzymologie), Génome bactérien (MeSH), Mixed function oxygenases (MeSH), Oxydoréduction (MeSH), Protéines de liaison à l'ADN (métabolisme), Protéines proto-oncogènes (métabolisme), Spécificité du substrat (MeSH).
- MESH :
English descriptors
- KwdEn :
- 5-Methylcytosine (metabolism), DNA Modification Methylases (metabolism), DNA-Binding Proteins (metabolism), Escherichia coli (enzymology), Genome, Bacterial (MeSH), Kinetics (MeSH), Mixed Function Oxygenases (MeSH), Oxidation-Reduction (MeSH), Proto-Oncogene Proteins (metabolism), Sequence Analysis, DNA (MeSH), Substrate Specificity (MeSH).
- MESH :
- chemical , metabolism : 5-Methylcytosine, DNA Modification Methylases, DNA-Binding Proteins, Proto-Oncogene Proteins.
- enzymology : Escherichia coli.
- Genome, Bacterial, Kinetics, Mixed Function Oxygenases, Oxidation-Reduction, Sequence Analysis, DNA, Substrate Specificity.
Abstract
BACKGROUND
DNA methylation serves as an important epigenetic mark in both eukaryotic and prokaryotic organisms. In eukaryotes, the most common epigenetic mark is 5-methylcytosine, whereas prokaryotes can have 6-methyladenine, 4-methylcytosine, or 5-methylcytosine. Single-molecule, real-time sequencing is capable of directly detecting all three types of modified bases. However, the kinetic signature of 5-methylcytosine is subtle, which presents a challenge for detection. We investigated whether conversion of 5-methylcytosine to 5-carboxylcytosine using the enzyme Tet1 would enhance the kinetic signature, thereby improving detection.
RESULTS
We characterized the kinetic signatures of various cytosine modifications, demonstrating that 5-carboxylcytosine has a larger impact on the local polymerase rate than 5-methylcytosine. Using Tet1-mediated conversion, we show improved detection of 5-methylcytosine using in vitro methylated templates and apply the method to the characterization of 5-methylcytosine sites in the genomes of Escherichia coli MG1655 and Bacillus halodurans C-125.
CONCLUSIONS
We have developed a method for the enhancement of directly detecting 5-methylcytosine during single-molecule, real-time sequencing. Using Tet1 to convert 5-methylcytosine to 5-carboxylcytosine improves the detection rate of this important epigenetic marker, thereby complementing the set of readily detectable microbial base modifications, and enhancing the ability to interrogate eukaryotic epigenetic markers.
DOI: 10.1186/1741-7007-11-4
PubMed: 23339471
PubMed Central: PMC3598637
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pubmed:23339471Le document en format XML
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<term>Escherichia coli (enzymology)</term>
<term>Genome, Bacterial (MeSH)</term>
<term>Kinetics (MeSH)</term>
<term>Mixed Function Oxygenases (MeSH)</term>
<term>Oxidation-Reduction (MeSH)</term>
<term>Proto-Oncogene Proteins (metabolism)</term>
<term>Sequence Analysis, DNA (MeSH)</term>
<term>Substrate Specificity (MeSH)</term>
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<term>Analyse de séquence d'ADN (MeSH)</term>
<term>Cinétique (MeSH)</term>
<term>DNA modification methylases (métabolisme)</term>
<term>Escherichia coli (enzymologie)</term>
<term>Génome bactérien (MeSH)</term>
<term>Mixed function oxygenases (MeSH)</term>
<term>Oxydoréduction (MeSH)</term>
<term>Protéines de liaison à l'ADN (métabolisme)</term>
<term>Protéines proto-oncogènes (métabolisme)</term>
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<term>Proto-Oncogene Proteins</term>
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<keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Escherichia coli</term>
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<keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>5-Méthyl-cytosine</term>
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<term>Protéines de liaison à l'ADN</term>
<term>Protéines proto-oncogènes</term>
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<term>Kinetics</term>
<term>Mixed Function Oxygenases</term>
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<term>Substrate Specificity</term>
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<front><div type="abstract" xml:lang="en"><p><b>BACKGROUND</b>
</p>
<p>DNA methylation serves as an important epigenetic mark in both eukaryotic and prokaryotic organisms. In eukaryotes, the most common epigenetic mark is 5-methylcytosine, whereas prokaryotes can have 6-methyladenine, 4-methylcytosine, or 5-methylcytosine. Single-molecule, real-time sequencing is capable of directly detecting all three types of modified bases. However, the kinetic signature of 5-methylcytosine is subtle, which presents a challenge for detection. We investigated whether conversion of 5-methylcytosine to 5-carboxylcytosine using the enzyme Tet1 would enhance the kinetic signature, thereby improving detection.</p>
</div>
<div type="abstract" xml:lang="en"><p><b>RESULTS</b>
</p>
<p>We characterized the kinetic signatures of various cytosine modifications, demonstrating that 5-carboxylcytosine has a larger impact on the local polymerase rate than 5-methylcytosine. Using Tet1-mediated conversion, we show improved detection of 5-methylcytosine using in vitro methylated templates and apply the method to the characterization of 5-methylcytosine sites in the genomes of Escherichia coli MG1655 and Bacillus halodurans C-125.</p>
</div>
<div type="abstract" xml:lang="en"><p><b>CONCLUSIONS</b>
</p>
<p>We have developed a method for the enhancement of directly detecting 5-methylcytosine during single-molecule, real-time sequencing. Using Tet1 to convert 5-methylcytosine to 5-carboxylcytosine improves the detection rate of this important epigenetic marker, thereby complementing the set of readily detectable microbial base modifications, and enhancing the ability to interrogate eukaryotic epigenetic markers.</p>
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<Abstract><AbstractText Label="BACKGROUND" NlmCategory="BACKGROUND">DNA methylation serves as an important epigenetic mark in both eukaryotic and prokaryotic organisms. In eukaryotes, the most common epigenetic mark is 5-methylcytosine, whereas prokaryotes can have 6-methyladenine, 4-methylcytosine, or 5-methylcytosine. Single-molecule, real-time sequencing is capable of directly detecting all three types of modified bases. However, the kinetic signature of 5-methylcytosine is subtle, which presents a challenge for detection. We investigated whether conversion of 5-methylcytosine to 5-carboxylcytosine using the enzyme Tet1 would enhance the kinetic signature, thereby improving detection.</AbstractText>
<AbstractText Label="RESULTS" NlmCategory="RESULTS">We characterized the kinetic signatures of various cytosine modifications, demonstrating that 5-carboxylcytosine has a larger impact on the local polymerase rate than 5-methylcytosine. Using Tet1-mediated conversion, we show improved detection of 5-methylcytosine using in vitro methylated templates and apply the method to the characterization of 5-methylcytosine sites in the genomes of Escherichia coli MG1655 and Bacillus halodurans C-125.</AbstractText>
<AbstractText Label="CONCLUSIONS" NlmCategory="CONCLUSIONS">We have developed a method for the enhancement of directly detecting 5-methylcytosine during single-molecule, real-time sequencing. Using Tet1 to convert 5-methylcytosine to 5-carboxylcytosine improves the detection rate of this important epigenetic marker, thereby complementing the set of readily detectable microbial base modifications, and enhancing the ability to interrogate eukaryotic epigenetic markers.</AbstractText>
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<Reference><Citation>Science. 2009 May 15;324(5929):930-5</Citation>
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