HPLC analysis of plant DNA methylation: a study of critical methodological factors

Johnston, Jason W.; Harding, Keith; Bremner, David H.; Souch, Graham; Green, Jon; Lynch, P. T.; Grout, B. W. W.; Benson, Erica E.

doi:10.1016/j.plaphy.2005.07.015

Cited by 64 publications

(42 citation statements)

References 26 publications

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“…Chromatographic assessment of global DNA methylation RP-HPLC quantification of overall DNA methylation was performed according to the procedure of Johnston et al (2005) using Waters 625 LC System. Briefly, the 4u Max-RP C12 (250 9 4.6 mm, Phenomenex) column combined with 4u Max-RP C12 pre-column were applied using linear gradient of the eluent A comprising of 0.5 % methanol plus 10 mM KH 2 PO 4 at a pH of 3.7 (v/v) and eluent B comprising of 10 % methanol in 10 mM KH 2 PO 4 , pH 3.7 (v/v) that changed from 100 % of A to 100 % of B within 10 min following 15 min of 100 % B eluent and ending with 100 % of the A eluent for 5 min at a flow rate equal to 1 ml min -1 .…”

Section: Rp-hplc Analysismentioning

confidence: 99%

DNA methylation changes in triticale due to in vitro culture plant regeneration and consecutive reproduction

Machczyńska

Orłowska

Mańkowski

et al. 2014

Plant Cell Tiss Organ Cult

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Doubled haploids of triticale are of interest for plant breeders due to hybrid breeding programs based on cytoplasmic male sterility Tt phenomenon. However, (epi)mutations appearing during in vitro culture regeneration may lead to a phenotypic variation that makes the uniformity of plant materials questionable. Using RP-HPLC genomic DNA methylation of donor doubled haploid plants utilized as a source of tissues for the in vitro regeneration (via androgenesis and somatic embryogenesis) of triticale cv. Bogo and their consecutive generative progeny was evaluated. It was demonstrated that in vitro cultures induced a decrease of the DNA methylation of the regenerants independently of the approach used for plant regeneration. The decrease in DNA methylation of genomic DNA proceeded up to the first/second successive generations followed by the beginning of its reestablishment. Moreover, somatic embryogenesis resulted in a higher level of genomic DNA demethylation in regenerants than androgenesis and the process of methylation seems to be affected by donor plant. It is being speculated that long term changes in genomic DNA methylation may be a source of off-type individuals that may spontaneously arise during plant breeding.

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Section: Rp-hplc Analysismentioning

confidence: 99%

DNA methylation changes in triticale due to in vitro culture plant regeneration and consecutive reproduction

Machczyńska

Orłowska

Mańkowski

et al. 2014

Plant Cell Tiss Organ Cult

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“…[29,38] The DNA samples were initially hydrolysed to 2′-deoxynucleoside 3′-monophosphates by an overnight incubation with a combination of an endoexonuclease (micrococcal nuclease) and 5′-exonuclease (calf spleen phosphodiesterase). The 2′-deoxynucleosides were generated by cleavage of the phosphate group following incubation with a 3′-nucleotidase (nuclease P1) for 4 h.…”

Section: Calculation Of the Percentage 5-medc In Dnamentioning

confidence: 99%

“…In principle the HPLC-UV approach used was similar to previously published methods. [25][26][27][28][29] …”

Section: Calculation Of the Percentage 5-medc In Dnamentioning

confidence: 99%

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Determination of 5-methyl-2′-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection

Sandhu

Kaur

Armstrong

et al. 2009

Journal of Chromatography B

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2 Abbreviations: 5-MedC, 5-methyl-2′-deoxycytidine; dC, 2′-deoxycytidine; dG, 2′-deoxyguanosine; T, thymidine; dA, 2′-deoxyadenosine; G, guanosine 3 AbstractThe formation of 5-methyl-2′-deoxycytidine (5-MedC) following methylation of the C-5 position of cytosine in genomic DNA provides an epigenetic mechanism for the regulation of gene expression and cellular differentiation. We describe the development of a method using HPLC-ultraviolet (UV) detection for the accurate determination of 5-MedC in DNA. Genomic DNA was obtained from HeLa cells and rat liver tissue using an optimised anion-exchange column DNA extraction procedure incorporating a ribonuclease incubation step to remove any potential interference from RNA. Following extraction the DNA samples were enzymatically hydrolysed to 2′-deoxynucleosides using a combination of an endo-exonuclease plus 5′-exonuclease together with a 3′-nucleotidase. The hydrolysed DNA samples (10 µg on column)were analysed using narrow-bore reverse phase HPLC-UV detection. The level of 5-MedC in the DNA samples was expressed as a percentage of the level of 2′-deoxycytidine (dC) determined from calibration lines constructed using authentic standards for 5-MedC and dC. The percentage 5-MedC level determined for commercially available calf thymus DNA was 6.26%, for HeLa cell DNA was 3.02% and for rat liver DNA was 3.55%.4

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“…Nucleic acids digestion procedures were based on the method described by Johnston et al (2005) and Fraga et al (2012). High performance liquid chromatography (HPLC) analysis was performed according to Johnston et al (2005).…”

Section: Plant Materialsmentioning

confidence: 99%

<b>Dynamics in global DNA methylation and endogenous polyamine levels during protocorm-like bodies induction of <i>Cattleya tigrina</i> A. Richard

Almeida

Fraga

Navarro

et al. 2017

Acta Sci. Biol. Sci.

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ABSTRACT. Cattleya tigrina is endemic to the Atlantic forest biome and classified as vulnerable in the Red Book of Brazilian Flora. In vitro techniques comprise valuable tools for the conservation of endangered plant species. The aim of the present study was to evaluate the morphological features, global DNA methylation levels and free polyamines during protocorm-like bodies (PLBs) induction of C. tigrina. Along with that, an efficient protocol for in vitro propagation of this species is proposed. The first evidences of PLBs induction in C. tigrina occurred at seven days in culture, starting from the basal portion of the leaf abaxial surface. A hypomethylation marked the beginning of cell differentiation, followed by an increased global DNA methylation at 35 days in culture, coinciding with a subtle change in the structures morphogenetic development. During PLBs induction, putrescine exhibited higher levels as compared to spermidine and spermine, and apparently presents a major role during the PLBs induction in C. tigrina. Due to the apparent secondary PLBs formation, this protocol can represent a highly efficient method for in vitro propagation of this species.Keywords: orchids, micropropagation, in vitro culture, epigenetics, PLBs.Dinâmica da metilação do DNA global e níveis endógenos de poliaminas durante a indução de estruturas semelhantes a protocormos de Cattleya tigrina A. Richard RESUMO. Cattleya tigrina é uma espécie endêmica do bioma Mata Atlântica e classificada como vulnerável no Livro Vermelho da Flora Brasileira. As técnicas in vitro compreendem ferramentas valiosas a serem empregadas na conservação de espécies de plantas ameaçadas. O objetivo do presente estudo foi avaliar as características morfológicas, os níveis globais de metilação do DNA e as poliaminas livres durante a indução de estruturas semelhantes a protocormos (ESPs). Paralelamente, um protocolo eficiente para a propagação in vitro desta espécie é apresentado. As primeiras evidências de indução de ESPs em C. tigrina foram observadas aos sete dias de cultivo, a partir da porção basal da superfície abaxial da folha. Uma hipometilação foi observada concomitante ao início da diferenciação celular, e um aumento da metilação global do DNA foi encontrada aos 35 dias de cultivo, coincidindo com uma sutil mudança no desenvolvimento morfogenético das estruturas. Durante a indução de ESPs, a putrescina exibiu níveis aumentados em comparação a espermidina e espermina e, aparentemente, apresenta um papel importante durante a indução dessas estruturas em C. tigrina. Devido à aparente formação secundária de ESPs, este protocolo pode representar um método altamente eficiente para a propagação in vitro desta espécie.Palavras-chave: orquideas, micropropagação, cultivo in vitro, epigenética, ESPs.

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HPLC analysis of plant DNA methylation: a study of critical methodological factors

Cited by 64 publications

References 26 publications

DNA methylation changes in triticale due to in vitro culture plant regeneration and consecutive reproduction

DNA methylation changes in triticale due to in vitro culture plant regeneration and consecutive reproduction

Determination of 5-methyl-2′-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection

<b>Dynamics in global DNA methylation and endogenous polyamine levels during protocorm-like bodies induction of <i>Cattleya tigrina</i> A. Richard

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