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Highly sensitive and multiplexed analysis of CpG methylation at single-base resolution with ligation-based exponential amplification

DNA methylation is a primary epigenetic mechanism for transcriptional regulation during normal development and the occurrence of diseases, including cancers. DNA methylation has been increasingly utilized as a biomarker for cancer detection and differential diagnosis. Generally, one type of cancer i...

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Autores principales: Su, Fengxia, Wang, Limei, Sun, Yueying, Liu, Chenghui, Duan, Xinrui, Li, Zhengping
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Royal Society of Chemistry 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494546/
https://www.ncbi.nlm.nih.gov/pubmed/28706642
http://dx.doi.org/10.1039/c4sc03135k
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author Su, Fengxia
Wang, Limei
Sun, Yueying
Liu, Chenghui
Duan, Xinrui
Li, Zhengping
author_facet Su, Fengxia
Wang, Limei
Sun, Yueying
Liu, Chenghui
Duan, Xinrui
Li, Zhengping
author_sort Su, Fengxia
collection PubMed
description DNA methylation is a primary epigenetic mechanism for transcriptional regulation during normal development and the occurrence of diseases, including cancers. DNA methylation has been increasingly utilized as a biomarker for cancer detection and differential diagnosis. Generally, one type of cancer is associated with several CpG methylation sites and detection of multiplexed CpG methylation can greatly improve the accuracy of cancer diagnosis. In this paper, we have developed a novel ligase chain reaction (LCR)-based method for multiplexed detection of CpG methylation in genomic DNA at single-base resolution. By rationally designing the two pairs of DNA probes for LCR, the bisulfite-treated methylated DNA target can be exponentially amplified by thermal cycling of the ligation reaction, in which one-base mismatch can be discriminated against, and thus high sensitivity and specificity for the detection of DNA methylation can be achieved. The LCR-based method can accurately determine as low as 10 aM methylated DNA fragment and 10 ng methylated genomic DNA. 0.1% methylated DNA can be detected in the presence of a large excess of unmethylated DNA. Moreover, by simply encoding one of the DNA probes in the LCR with a different length of poly(A) for detection of methylation at different CpG sites, the CpG methylation at different sites can produce LCR products with different lengths, and thus, can be simultaneously detected with one-tube LCR amplification and separation by capillary electrophoresis.
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spelling pubmed-54945462017-07-13 Highly sensitive and multiplexed analysis of CpG methylation at single-base resolution with ligation-based exponential amplification Su, Fengxia Wang, Limei Sun, Yueying Liu, Chenghui Duan, Xinrui Li, Zhengping Chem Sci Chemistry DNA methylation is a primary epigenetic mechanism for transcriptional regulation during normal development and the occurrence of diseases, including cancers. DNA methylation has been increasingly utilized as a biomarker for cancer detection and differential diagnosis. Generally, one type of cancer is associated with several CpG methylation sites and detection of multiplexed CpG methylation can greatly improve the accuracy of cancer diagnosis. In this paper, we have developed a novel ligase chain reaction (LCR)-based method for multiplexed detection of CpG methylation in genomic DNA at single-base resolution. By rationally designing the two pairs of DNA probes for LCR, the bisulfite-treated methylated DNA target can be exponentially amplified by thermal cycling of the ligation reaction, in which one-base mismatch can be discriminated against, and thus high sensitivity and specificity for the detection of DNA methylation can be achieved. The LCR-based method can accurately determine as low as 10 aM methylated DNA fragment and 10 ng methylated genomic DNA. 0.1% methylated DNA can be detected in the presence of a large excess of unmethylated DNA. Moreover, by simply encoding one of the DNA probes in the LCR with a different length of poly(A) for detection of methylation at different CpG sites, the CpG methylation at different sites can produce LCR products with different lengths, and thus, can be simultaneously detected with one-tube LCR amplification and separation by capillary electrophoresis. Royal Society of Chemistry 2015-03-01 2014-12-11 /pmc/articles/PMC5494546/ /pubmed/28706642 http://dx.doi.org/10.1039/c4sc03135k Text en This journal is © The Royal Society of Chemistry 2014 http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 Unported License (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Chemistry
Su, Fengxia
Wang, Limei
Sun, Yueying
Liu, Chenghui
Duan, Xinrui
Li, Zhengping
Highly sensitive and multiplexed analysis of CpG methylation at single-base resolution with ligation-based exponential amplification
title Highly sensitive and multiplexed analysis of CpG methylation at single-base resolution with ligation-based exponential amplification
title_full Highly sensitive and multiplexed analysis of CpG methylation at single-base resolution with ligation-based exponential amplification
title_fullStr Highly sensitive and multiplexed analysis of CpG methylation at single-base resolution with ligation-based exponential amplification
title_full_unstemmed Highly sensitive and multiplexed analysis of CpG methylation at single-base resolution with ligation-based exponential amplification
title_short Highly sensitive and multiplexed analysis of CpG methylation at single-base resolution with ligation-based exponential amplification
title_sort highly sensitive and multiplexed analysis of cpg methylation at single-base resolution with ligation-based exponential amplification
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494546/
https://www.ncbi.nlm.nih.gov/pubmed/28706642
http://dx.doi.org/10.1039/c4sc03135k
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