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A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements
- Source :
- Web of Science, Repositório Institucional da UNESP, Universidade Estadual Paulista (UNESP), instacron:UNESP, Scopus-Elsevier
- Publication Year :
- 2004
- Publisher :
- Oxford University Press, 2004.
-
Abstract
- Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:18:57Z No. of bitstreams: 1 WOS000220490400055.pdf: 202398 bytes, checksum: 84d383a3383809a2100fdc655a9d1888 (MD5) Made available in DSpace on 2014-02-26T17:18:57Z (GMT). No. of bitstreams: 1 WOS000220490400055.pdf: 202398 bytes, checksum: 84d383a3383809a2100fdc655a9d1888 (MD5) Previous issue date: 2004-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:00:18Z No. of bitstreams: 1 WOS000220490400055.pdf: 202398 bytes, checksum: 84d383a3383809a2100fdc655a9d1888 (MD5) Made available in DSpace on 2014-05-20T14:00:18Z (GMT). No. of bitstreams: 1 WOS000220490400055.pdf: 202398 bytes, checksum: 84d383a3383809a2100fdc655a9d1888 (MD5) Previous issue date: 2004-02-01 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. Univ Texas, MD Anderson Canc Ctr, Dept Leukemia, Houston, TX 77030 USA UNESP, IBILCE, Dept Biol, São Paulo, Brazil UNESP, IBILCE, Dept Biol, São Paulo, Brazil
- Subjects :
- Bisulfite sequencing
Alu element
Biology
Decitabine
Polymerase Chain Reaction
Sensitivity and Specificity
chemistry.chemical_compound
Alu Elements
Cell Line, Tumor
Genetics
Humans
Sulfites
Methylated DNA immunoprecipitation
NAR Methods Online
Repetitive Sequences, Nucleic Acid
Reproducibility of Results
Methylation
DNA
Sequence Analysis, DNA
DNA Methylation
Molecular biology
Long Interspersed Nucleotide Elements
chemistry
CpG site
DNA methylation
Mutation
Azacitidine
Illumina Methylation Assay
CpG Islands
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Web of Science, Repositório Institucional da UNESP, Universidade Estadual Paulista (UNESP), instacron:UNESP, Scopus-Elsevier
- Accession number :
- edsair.doi.dedup.....20dd44fb389694aa885049deff808ad3