Determination of the DNA methylation level of the marbled crayfish: An increase in sample throughput by an optimised sample preparation

Production of different phenotypes from the same genotype in the same environment by developmental variation

“…Analyses were performed with capillary electrophoresis, using a novel and highly sensitive sample preparation technique (Schiewek et al, 2007). Each sample was measured at least 20 times.…”

“…In order to test if differently grown batch-mates of the marbled crayfish also show differences in the epigenetic code, we measured global methylation of cytosines in the DNA of the hepatopancreas and the abdominal musculature, using an improved and highly sensitive analytical technique recently developed by us (Schiewek et al, 2007). This method allows the determination of very low genome-wide methylation levels (<1%) from DNA samples as small as 1·g.…”

Suitability of the clonal marbled crayfish for biogerontological research: a review and perspective, with remarks on some further crustaceans

“…However, not all of the established ageing models are suitable to investigate alterations of DNA methylation throughout life because Caenorhabditis elegans lacks methylated DNA at all (Bird 2002) and Drosophila melanogaster possesses methylated DNA only in the early stages of embryonic development (Lyko et al 2006). The marbled crayfish, in contrast, has methylated DNA in both the juvenile and adult life stages, corresponding to approximately half of the value of humans (Schiewek et al 2007;Vogt et al 2008). Measurement of global 5-methyl cytosine methylation in two communally reared batches of the marbled crayfish revealed variations among batch-mates, tissue and age (Vogt et al 2008).…”

“…Alterations of global DNA methylation during lifetime could be determined from blood or limb muscle samples using a refined analytical technique to precisely measure low methylation levels in small sample sizes (Schiewek et al 2007). Age-related changes of the genome-wide DNA methylation patterns in various tissues could be determined by restriction landmark genomic scanning even without knowing particular genes (Thompson et al 2010).…”

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

“…Nonchromatographic methods used for the assessment of genomic DNA methylation include: the preferential conversion of unmethylated cytosine to uracil by sodium bisulphite and subsequent determination by PCR amplification and sequencing [2,8]; a radioactive assay which involves the de novo transfer of a radioactive methyl donor by DNA methyltransferase to 2′-deoxycytidine (dC) sites within the DNA [9]; a Southern blot assay that utilises DNA digestion with restriction endonucleases that are sensitive to methylated sites [2,10]; an assay involving single nucleotide extension with radiolabelled [ 3 H]dCTP following cleavage of DNA with methylation-sensitive restriction endonucleases [11]. Chromatographic approaches include methods such as thin layer chromatography (following 32 P-postlabelling) [12,13], high performance liquid chromatography (HPLC) coupled to UV and mass spectrometric [14][15][16][17] detection, gas chromatography [18,19] and capillary electrophoresis [20][21][22][23][24].…”