Idioma:

Specific Method for the Determination of Genomic DNA Methylation by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

Song, Liguo ; James, Smitha R ; Kazim, Latif ; Karpf, Adam R

Analytical chemistry (Washington), 2005-01, Vol.77 (2), p.504-510 [Periódico revisado por pares]

Washington, DC: American Chemical Society

Texto completo disponível

Citações Citado por

Enviar para

Título:
Specific Method for the Determination of Genomic DNA Methylation by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry
Autor: Song, Liguo ; James, Smitha R ; Kazim, Latif ; Karpf, Adam R
Assuntos: Analytical chemistry ; Cell Line, Tumor ; Chemistry ; Chromatographic methods and physical methods associated with chromatography ; Chromatography, Liquid - methods ; Deoxycytidine - analogs & derivatives ; Deoxycytidine - analysis ; Deoxyribonucleic acid ; DNA ; DNA Methylation ; Enzymes ; Exact sciences and technology ; Genome - drug effects ; Humans ; Other chromatographic methods ; Reproducibility of Results ; Sensitivity and Specificity ; Spectrometric and optical methods ; Spectrometry, Mass, Electrospray Ionization - methods ; Spectrum analysis ; Tandem Mass Spectrometry - methods
É parte de: Analytical chemistry (Washington), 2005-01, Vol.77 (2), p.504-510
Notas: ark:/67375/TPS-N4HN7RJ6-T
istex:A2661AE08A7DA47ABAA39D7730F685A5337F2F64
ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
Descrição: Herein we report a novel method for determining genomic DNA methylation that utilizes liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to measure 5-methyl-2‘-deoxycytidine levels following enzymatic hydrolysis of genomic DNA. LC separation of 5-methyl-2‘-deoxycytidine from the four deoxyribonucleosides, the four ribonucleosides, and 5-methyl-2‘-cytidine, a RNA methylation product, has been achieved within 15 min. In combination with ESI-MS/MS detection, the reported method is highly specific and extremely sensitive with a limit of detection (LOD) of 0.2 fmol and a quantification linearity range from 1 fmol to 20 pmol. Genomic DNA methylation was expressed as the ratio of 5-methyl-2‘-deoxycytidine to 2‘-deoxyguanosine and was determined directly using 2‘-deoxyguanosine as the internal standard. Because deoxycytidine methylation typically ranges from 2 to 6% in mammalian genomes, and pharmacological or genetic manipulations have not achieved levels lower than 0.1%, we validated the assay for methylation levels ranging from 0.05 to 10%. Importantly, both RNA contamination and incomplete DNA hydrolysis had no appreciable effect on 5-methyl-2‘-deoxycytidine quantification. LOD studies indicate that only 4 ng of DNA is required for this assay. This LOD should permit the use of this method for applications having limiting amounts of DNA that were not previously candidates for global genomic DNA methylation analysis, e.g., clinical trial samples, or cells collected by laser capture microdissection.
Editor: Washington, DC: American Chemical Society
Idioma: Inglês

Links

Voltar para lista de resultados

Anterior Resultado 6 Avançar Ir para próxima página

Realização: Logos de Redes Sociais:

Specific Method for the Determination of Genomic DNA Methylation by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

Song, Liguo ; James, Smitha R ; Kazim, Latif ; Karpf, Adam R

Washington, DC: American Chemical Society

Buscando em bases de dados remotas. Favor aguardar.