Sensitive detection of 5-methylcytosine and quantitation of the 5-methylcytosine/cytosine ratio in DNA by gas chromatography-mass spectrometry using multiple specific ion monitoring

Singer, Judith A.; Schnute, William C.; Shively, John E.; Todd, Charles W.; Riggs, Arthur D.

doi:10.1016/0003-2697(79)90363-4

Cited by 46 publications

(27 citation statements)

References 7 publications

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“…in Drosophila DNA 3 highly sensitive methods were used: high resolution mass spectrometry [2], gas chromatography-mass spectrometry [3] and high-performance liquid chromatography [4]. In none of these methods could m5Cyt be detected (not shown).…”

Section: Resultsmentioning

confidence: 99%

The absence of detectable methylated bases in Drosophila melanogaster DNA

et al. 1982

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Section: Resultsmentioning

confidence: 99%

The absence of detectable methylated bases in Drosophila melanogaster DNA

et al. 1982

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“…The m5C contents in the DNA fractions and the isolated restriction fragments were determined after hydrolysis with 98% formic acid by thin-layer chromatography on cellulose, by gas chromatography, and by combined gas chromatography and mass spectrometry (18,19). Sample injection was as described by Ros (20).…”

Section: Temperature Ocmentioning

confidence: 99%

Sequence arrangement of a highly methylated satellite DNA of a plant, Scilla : A tandemly repeated inverted repeat

Deumling

1981

Proc. Natl. Acad. Sci. U.S.A.

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G+C-rich satellite DNA, representing about 19% of total nuclear DNA, was isolated from various tissues of the MOnocotyledonousant, ScitasibeDica, byusing A+-Cs SO4gra-diemnot tedonou is satellite DNA had an unusi hi melting point and a high methylcytosine (m5C) content (-25% of total bases; m5C/cytosine ratio 1L.5) and was localized, by in situ hybridization, in the heterochromatin regions of the chromosomes. Digestion with restriction endonuclease Hae m yielded a series of fragments rangingfrom 35 to several hundred nucleotide pairs. The major fragments, I-IV (35, 50, 59, and 69, nucleotide pairs, respectively), were isolated, and their nucleotide sequences were determined. The dominant fragment I was a highly symmetrical molecule, with a basically palindromic arrangement. This sequence represented the basic unit of Scilla satellite DNA and was tandemly repeated many times, with some base substitutions and multiple successive insertions of the tetranucleotide G-T-C-C. The dinucleotide CpG was the commonest nearest-neighbor sequence. Thin layer chromatography, DNA sequence analysis, and gas chromatography combined with mass spectrometry showed the high m5C content (m5C/Cyt = 2.2 and 2.8, respectively, for fragments II and IEl). Identical cleavage fragments were found in satellite DNAs from two other species of this genus (S. amoena and S. ingridae), which suggests that this constitutively methylated sequence is evolutionarily stable. The sequence arrangement of this plant satellite DNA is compared with those reported for several animal satellite DNAs. Temperature, OCThe nuclear DNA of most, probably all, eukaryotic cells contains fractions of highly reiterated, relatively simple sequences that, in many species, appear as DNA satellites separable from the bulk of the DNA (1). In a variety of animals, the nucleotide sequence arrangement of such satellite DNAs has been elucidated; as a general principle, they contain tandem repeats of a specific sequence unit, which is different in composition and size in different organisms (1-7). Satellite DNAs have also been described (8-11) in several plants that are enriched in heterochromatin. Among the plants that show conspicuous heterochromatin arrays, the monocotyledonous bluebell species, Scilla siberica, has been studied in detail (9,10,(12)(13)(14). The satellite DNA in the heterochromatin of this plant (9, 10) has been analyzed, and the nucleotide sequence of a part of it has been determined. The results show some unusual features ofsatellite DNA organization-multiple tandem arrays of an inverted repeat, multiple insertions of a tetranucleotide, and remarkably high contents of 5-methylcytosine (m5C)

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“…GC-MS permits the avoidance of resolution limitation by coupling elution time to mass spectrum information. It also allows more selective and sensitive analysis than the above instruments alone, allowing the analysis of the methylated base in a small amount of DNA [24]. Moreover, ideal internal standards choice can improve GC-MS quantitation measurements.…”

mentioning

confidence: 99%

“…Moreover, ideal internal standards choice can improve GC-MS quantitation measurements. A method able to estimate DNA methylation percentage as 5mCyt/total Cyt would have both 5mCyt and Cyt measurements as near as possible to the real values [1,[23][24][25]. Since 5mCyt levels are much lower than Cyt levels, it is necessary to employ two different internal standards to guarantee a correct quantification of the two molecules and to avoid errors in measurements.…”

mentioning

confidence: 99%