A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements
Autor(a) principal: | |
---|---|
Data de Publicação: | 2004 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1093/nar/gnh032 http://hdl.handle.net/11449/21329 |
Resumo: | We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1-16% decrease in Alu element and 18-60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation. |
id |
UNSP_714bb1699a674ee6edac7a529a4177e5 |
---|---|
oai_identifier_str |
oai:repositorio.unesp.br:11449/21329 |
network_acronym_str |
UNSP |
network_name_str |
Repositório Institucional da UNESP |
repository_id_str |
2946 |
spelling |
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elementsWe report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1-16% decrease in Alu element and 18-60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.Univ Texas, MD Anderson Canc Ctr, Dept Leukemia, Houston, TX 77030 USAUNESP, IBILCE, Dept Biol, São Paulo, BrazilUNESP, IBILCE, Dept Biol, São Paulo, BrazilOxford University PressUniv TexasUniversidade Estadual Paulista (Unesp)Yang, A. S.Estecio, MRHDoshi, K.Kondo, Y.Tajara, E. H.Issa, JPJ2014-05-20T14:00:18Z2014-05-20T14:00:18Z2004-02-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article11application/pdfhttp://dx.doi.org/10.1093/nar/gnh032Nucleic Acids Research. Oxford: Oxford Univ Press, v. 32, n. 3, 11 p., 2004.0305-1048http://hdl.handle.net/11449/2132910.1093/nar/gnh032WOS:000220490400055WOS000220490400055.pdfWeb of Sciencereponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengNucleic Acids Research11.5619,025info:eu-repo/semantics/openAccess2023-10-19T06:08:26Zoai:repositorio.unesp.br:11449/21329Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T15:22:08.045691Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements |
title |
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements |
spellingShingle |
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements Yang, A. S. |
title_short |
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements |
title_full |
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements |
title_fullStr |
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements |
title_full_unstemmed |
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements |
title_sort |
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements |
author |
Yang, A. S. |
author_facet |
Yang, A. S. Estecio, MRH Doshi, K. Kondo, Y. Tajara, E. H. Issa, JPJ |
author_role |
author |
author2 |
Estecio, MRH Doshi, K. Kondo, Y. Tajara, E. H. Issa, JPJ |
author2_role |
author author author author author |
dc.contributor.none.fl_str_mv |
Univ Texas Universidade Estadual Paulista (Unesp) |
dc.contributor.author.fl_str_mv |
Yang, A. S. Estecio, MRH Doshi, K. Kondo, Y. Tajara, E. H. Issa, JPJ |
description |
We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1-16% decrease in Alu element and 18-60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation. |
publishDate |
2004 |
dc.date.none.fl_str_mv |
2004-02-01 2014-05-20T14:00:18Z 2014-05-20T14:00:18Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1093/nar/gnh032 Nucleic Acids Research. Oxford: Oxford Univ Press, v. 32, n. 3, 11 p., 2004. 0305-1048 http://hdl.handle.net/11449/21329 10.1093/nar/gnh032 WOS:000220490400055 WOS000220490400055.pdf |
url |
http://dx.doi.org/10.1093/nar/gnh032 http://hdl.handle.net/11449/21329 |
identifier_str_mv |
Nucleic Acids Research. Oxford: Oxford Univ Press, v. 32, n. 3, 11 p., 2004. 0305-1048 10.1093/nar/gnh032 WOS:000220490400055 WOS000220490400055.pdf |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Nucleic Acids Research 11.561 9,025 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
11 application/pdf |
dc.publisher.none.fl_str_mv |
Oxford University Press |
publisher.none.fl_str_mv |
Oxford University Press |
dc.source.none.fl_str_mv |
Web of Science reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1808128502925361152 |