A sensitive immunochemical method for detecting 5mC in DNA fragments

Achwal, CW; Chandra, H. Sharat

doi:10.1016/0014-5793(82)80791-6

Cited by 19 publications

(17 citation statements)

References 22 publications

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“…For example, if D. mefanogaster DNA contains at least one 5mC residue in 50000 nucleotides a positive reaction would be seen in this immunochemical method if the total amount of DNA in the spot is 1100 ng. This conclusion follows from our earlier finding that these antibodies are capable of detecting 0.005 pmol 5mC in 10 ng &Yl74 DNA [15]. This level of methylation, it appears, can be missed when less sensitive methods are employed.…”

Section: Sensitivity Of the Antibody Methodssupporting

confidence: 60%

“…lb). As described in [15], the Ka for 5mC, was 1.56 x IO9 M -r. Competition assays were carried out by incubating the labelled antigen and antibody in the presence of competing unlabelled molecules. Adenosine and 2-methyladenine at 3.3 x 10 -' M did not inhibit the binding of 6mA antibodies to labelled 6mA.…”

Section: Methodsmentioning

confidence: 71%

“…in [15]. The blot was incubated for 4-6 h with one of the three antibodies, washed in Trisbuffered saline (TBS; 10 mM Tris-HQ (pH 7.5), 0.14 M NaCl) and treated with biotinylated antiantibody.…”

Section: Spot Assaymentioning

confidence: 99%

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Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA

1983

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We have reported that production and characterization of antibodies highly specific to 5-methyl-cytosine (SmC) and the development of a sensitive immunochemicai method for the detection of 5mC in DNA [EBBS Lett. (1982) 150, 4691. Extension of this method to two other modified bases, 6-m~hyladenine (6mA) and %methyiguanine (7mG), is reported here. By use of this immunochemical approach, we are able to detect 5mC, 6mA and 7mG in human and Drosophila DNA and confirm their presence in the DNA of two mealybug species. DNA methylation Biotin-avidin cross-linking DNA-protein interaction Z-DNA X-chromosome inactivationGene regulation

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Section: Sensitivity Of the Antibody Methodssupporting

confidence: 60%

Section: Methodsmentioning

confidence: 71%

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Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA

1983

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“…Immunochemical assay for detection of 5mC in DNA was carried out according to Achwal and Chandra [21]. After the in vitro reaction, product DNA was reextracted with phenol-chloroform and precipitated with ethanol.…”

Section: In Vitro Assay For Methylasementioning

confidence: 99%

“…Immunochemical reactions using antibodies directed against 5mC or 6mA were carried out on these DNA samples, dissolved in 10 mM Tris-HC1, and 1 mM ED-TA pH 7.4. The DNA samples were spotted on nitrocellulose filters, fixed by baking in a vacuum oven and treated with antibodies [21]. The antibodies were raised in rabbits using B SA conjugates oof 5-methylcytidine or 6-methyladenine as antigens [21].…”

Section: In Vitro Assay For Methylasementioning

confidence: 99%

Detection of a CpA methylase in an insect system: characterization and substrate specificity

Devajyothi

Brahmachari

1992

Mol Cell Biochem

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A cytosine-specific DNA methyltransferase (EC 2.1.1.37) has been purified to near homogeneity from a mealybug (Planococcus lilacinus). The enzyme can methylate cytosine residues in CpG sequences as well as CpA sequences. The apparent molecular weight of the enzyme was estimated as 135,000 daltons by FPLC. The enzyme exhibits a processive mode of action and a salt dependence similar to mammalian methylases. Mealybug methylase exhibits a preference for denatured DNA substrates.

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Estimation of the amount of 5-methylcytosine in Drosophila melanogaster DNA by amplified ELISA and photoacoustic spectroscopy.

Achwal¹,

Ganguly²,

Chandra³

1984

The EMBO Journal

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We have previously reported a sensitive immunochemical method for detecting 5‐methylcytosine in DNA which involves spotting DNA samples on nitrocellulose paper and detection of 5‐methylcytosine, if any, by a combination of the double antibody method and a staining reaction brought about by biotin‐avidin and peroxidase. We report here a linear relationship between the concentration of 5‐methylcytosine in DNA and staining intensity, as recorded by photoacoustic spectroscopy. It appears possible to obtain, by this method, reliable quantitative estimates of 5‐methylcytosine in nanogram quantities of intact DNA. When Drosophila melanogaster DNA was assayed for the presence of 5‐methylcytosine by this method, a faint but clearly positive reaction was obtained. When the photoacoustic intensity of this stained spot is compared with a calibration plot derived from phi X174 DNA whose 5‐methylcytosine content is known, we obtain, for D. melanogaster DNA, one 5‐methylcytosine residue in approximately 12 500 bases or 0.008 mol% methylation.

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A sensitive immunochemical method for detecting 5mC in DNA fragments

Abstract: Modified nucleotides Southern transfer Biotin-avidin cross-linking X-chromosome inactivationGene expression

Cited by 19 publications

References 22 publications

Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA

Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA

Detection of a CpA methylase in an insect system: characterization and substrate specificity

Estimation of the amount of 5-methylcytosine in Drosophila melanogaster DNA by amplified ELISA and photoacoustic spectroscopy.

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