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Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input

The current methods for quantifying genome-wide 5-methylcytosine (5mC) oxides are still scarce, mostly restricted with two limitations: assay sensitivity is seriously compromised with cost, assay time and sample input; epigenetic information is irreproducible during polymerase chain reaction (PCR) a...

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Autores principales: Chen, Danping, Wang, Yang, Mo, Mingming, Zhang, Junjie, Zhang, Yanfei, Xu, Yuzhi, Liu, Si-Yang, Chen, Jun, Ma, Yingjun, Zhang, Li, Dai, Zong, Cai, Chun, Zou, Xiaoyong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821303/
https://www.ncbi.nlm.nih.gov/pubmed/31418020
http://dx.doi.org/10.1093/nar/gkz704
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author Chen, Danping
Wang, Yang
Mo, Mingming
Zhang, Junjie
Zhang, Yanfei
Xu, Yuzhi
Liu, Si-Yang
Chen, Jun
Ma, Yingjun
Zhang, Li
Dai, Zong
Cai, Chun
Zou, Xiaoyong
author_facet Chen, Danping
Wang, Yang
Mo, Mingming
Zhang, Junjie
Zhang, Yanfei
Xu, Yuzhi
Liu, Si-Yang
Chen, Jun
Ma, Yingjun
Zhang, Li
Dai, Zong
Cai, Chun
Zou, Xiaoyong
author_sort Chen, Danping
collection PubMed
description The current methods for quantifying genome-wide 5-methylcytosine (5mC) oxides are still scarce, mostly restricted with two limitations: assay sensitivity is seriously compromised with cost, assay time and sample input; epigenetic information is irreproducible during polymerase chain reaction (PCR) amplification without bisulfite pretreatment. Here, we propose a novel Polymerization Retardation Isothermal Amplification (PRIA) strategy to directly amplify the minute differences between epigenetic bases and others by arranging DNA polymerase to repetitively pass large electron-withdrawing groups tagged 5mC-oxides. We demonstrate that low abundant 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) in genomic DNA can be accurately quantified within 10 h with 100 ng sample input on a laboratory real-time quantitative PCR instrument, and even multiple samples can be analyzed simultaneously in microplates. The global levels of 5hmC and 5fC in mouse and human brain tissues, rat hippocampal neuronal tissue, mouse kidney tissue and mouse embryonic stem cells were quantified and the observations not only confirm the widespread presence of 5hmC and 5fC but also indicate their significant variation in different tissues and cells. The strategy is easily performed in almost all research and medical laboratories, and would provide the potential capability to other candidate modifications in nucleotides.
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spelling pubmed-68213032019-11-04 Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input Chen, Danping Wang, Yang Mo, Mingming Zhang, Junjie Zhang, Yanfei Xu, Yuzhi Liu, Si-Yang Chen, Jun Ma, Yingjun Zhang, Li Dai, Zong Cai, Chun Zou, Xiaoyong Nucleic Acids Res Methods Online The current methods for quantifying genome-wide 5-methylcytosine (5mC) oxides are still scarce, mostly restricted with two limitations: assay sensitivity is seriously compromised with cost, assay time and sample input; epigenetic information is irreproducible during polymerase chain reaction (PCR) amplification without bisulfite pretreatment. Here, we propose a novel Polymerization Retardation Isothermal Amplification (PRIA) strategy to directly amplify the minute differences between epigenetic bases and others by arranging DNA polymerase to repetitively pass large electron-withdrawing groups tagged 5mC-oxides. We demonstrate that low abundant 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) in genomic DNA can be accurately quantified within 10 h with 100 ng sample input on a laboratory real-time quantitative PCR instrument, and even multiple samples can be analyzed simultaneously in microplates. The global levels of 5hmC and 5fC in mouse and human brain tissues, rat hippocampal neuronal tissue, mouse kidney tissue and mouse embryonic stem cells were quantified and the observations not only confirm the widespread presence of 5hmC and 5fC but also indicate their significant variation in different tissues and cells. The strategy is easily performed in almost all research and medical laboratories, and would provide the potential capability to other candidate modifications in nucleotides. Oxford University Press 2019-11-04 2019-08-16 /pmc/articles/PMC6821303/ /pubmed/31418020 http://dx.doi.org/10.1093/nar/gkz704 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Chen, Danping
Wang, Yang
Mo, Mingming
Zhang, Junjie
Zhang, Yanfei
Xu, Yuzhi
Liu, Si-Yang
Chen, Jun
Ma, Yingjun
Zhang, Li
Dai, Zong
Cai, Chun
Zou, Xiaoyong
Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input
title Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input
title_full Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input
title_fullStr Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input
title_full_unstemmed Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input
title_short Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input
title_sort polymerization retardation isothermal amplification (pria): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821303/
https://www.ncbi.nlm.nih.gov/pubmed/31418020
http://dx.doi.org/10.1093/nar/gkz704
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