Fast and reliable method to estimate global DNA methylation in plants and fungi with high-pressure liquid chromatography (HPLC)-ultraviolet detection and even more sensitive one with HPLC-mass spectrometry.
J Biotechnol
; 374: 1-4, 2023 Sep 10.
Article
en En
| MEDLINE
| ID: mdl-37499875
DNA (Deoxyribonucleic acid) methylation is one of the epigenetic modifications of DNA, acting as a bridge between genotype and phenotype. Thus, disruption of DNA methylation pattern has tremendous consequences for organism development. Current methods to determine DNA methylation suffer from methodological drawbacks like high requirement of DNA and poor reproducibility of chromatograms. Here we provide a fast and reliable method using high-pressure liquid chromatography (HPLC)-ultraviolet (UV) detector and even more sensitive one with HPLC- mass spectrometry (MS) and we test this method with various plant and fungal DNA isolates. We optimized the preparation of the DNA degradation step to decrease background noise, we improved separation conditions to provide reliable and reproducible chromatograms and conditions to measure nucleotides in HPLC-MS. We showed that global DNA methylation level can be accurately and reproducibly measured with as little as 0.2 µM for HPLC-UV and 0.02 µM for HPLC-MS of methylated cytosine.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Metilación de ADN
/
Hongos
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
J Biotechnol
Asunto de la revista:
BIOTECNOLOGIA
Año:
2023
Tipo del documento:
Article
Pais de publicación:
Países Bajos