Rapid analysis of CpG methylation patterns using RNase T1 cleavage and MALDI-TOF | Zendy

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Rapid analysis of CpG methylation patterns using RNase T1 cleavage and MALDI-TOF

Author(s) -

P Schatz

Publication year - 2004

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/gnh165

Subject(s) - biology , microbiology and biotechnology , bisulfite sequencing , methylation , dna methylation , dna , bisulfite , cpg site , methylated dna immunoprecipitation , rnase p , rna , illumina methylation assay , rnase h , biochemistry , gene , gene expression

Here, we introduce a method for the fast and accurate analysis of DNA methylation based on bisulfite-treated DNA. The target region is PCR amplified using a T7 RNA polymerase promoter-tagged primer. A subsequent in vitro transcription leads to a transcript which contains guanosine residues only at sites that contained methylated cytosines before bisulfite treatment. In a single tube reaction using guanosine-specific cleavage by RNase T1, a specific pattern of RNA fragments is formed. This pattern directly represents the methylation state of the sample DNA and is analyzed using matrix-assisted laser desorption ionization time-of-flight technology. This method was successfully applied to the analysis of artificially methylated and unmethylated DNA, mixtures thereof and colon DNA samples. The applicability for the analysis of both PCR products and cloned PCR products is demonstrated. The observed methylation patterns were confirmed by bisulfite sequencing

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