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

被引：6

作者：

Adamczyk, Sylwia ^{[1
]}

Chojak-Kozniewska, Joanna ^{[2
]}

Poimala, Anna ^{[1
]}

Velmala, Sannakajsa ^{[1
]}

Adamczyk, Bartosz ^{[1
]}

机构：

[1] Nat Res Inst Finland Luke, Latokartanonkaari 9, Helsinki 00790, Finland

[2] Natl Res Inst, Plant Breeding & Acclimatizat Inst IHAR, PL-05870 Blonie, Poland

来源：

JOURNAL OF BIOTECHNOLOGY | 2023年 / 374卷

基金：

芬兰科学院;

关键词：

Chromatography; DNA methylation; Fungal DNA; Plant DNA;

D O I：

10.1016/j.jbiotec.2023.07.008

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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 & mu;M for HPLC-UV and 0.02 & mu;M for HPLC-MS of methylated cytosine.

引用

页码：1 / 4

页数：4

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