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. 1984 Feb;3(2):263–266. doi: 10.1002/j.1460-2075.1984.tb01795.x

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

C W Achwal, P Ganguly, H S Chandra
PMCID: PMC557333  PMID: 6232132

Abstract

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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Selected References

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  1. Achwal C. W., Chandra H. S. A sensitive immunochemical method for detecting 5mC in DNA fragments. FEBS Lett. 1982 Dec 27;150(2):469–472. doi: 10.1016/0014-5793(82)80791-6. [DOI] [PubMed] [Google Scholar]
  2. Achwal C. W., Iyer C. A., Chandra H. S. Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA. FEBS Lett. 1983 Jul 25;158(2):353–358. doi: 10.1016/0014-5793(83)80612-7. [DOI] [PubMed] [Google Scholar]
  3. Behe M., Zimmerman S., Felsenfeld G. Changes in the helical repeat of poly(dG-m5dC) . poly(dG-m5dC) and poly(dG-dC) . poly(dG-dC) associated with the B-Z transition. Nature. 1981 Sep 17;293(5829):233–235. doi: 10.1038/293233a0. [DOI] [PubMed] [Google Scholar]
  4. Doerfler W. DNA methylation and gene activity. Annu Rev Biochem. 1983;52:93–124. doi: 10.1146/annurev.bi.52.070183.000521. [DOI] [PubMed] [Google Scholar]
  5. Ehrlich M., Gama-Sosa M. A., Huang L. H., Midgett R. M., Kuo K. C., McCune R. A., Gehrke C. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells. Nucleic Acids Res. 1982 Apr 24;10(8):2709–2721. doi: 10.1093/nar/10.8.2709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ehrlich M., Wang R. Y. 5-Methylcytosine in eukaryotic DNA. Science. 1981 Jun 19;212(4501):1350–1357. doi: 10.1126/science.6262918. [DOI] [PubMed] [Google Scholar]
  7. Gruenbaum Y., Stein R., Cedar H., Razin A. Methylation of CpG sequences in eukaryotic DNA. FEBS Lett. 1981 Feb 9;124(1):67–71. doi: 10.1016/0014-5793(81)80055-5. [DOI] [PubMed] [Google Scholar]
  8. Holliday R., Pugh J. E. DNA modification mechanisms and gene activity during development. Science. 1975 Jan 24;187(4173):226–232. [PubMed] [Google Scholar]
  9. Hsu S. M., Raine L., Fanger H. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol. 1981 May;75(5):734–738. doi: 10.1093/ajcp/75.5.734. [DOI] [PubMed] [Google Scholar]
  10. Hsu S. M., Raine L., Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981 Apr;29(4):577–580. doi: 10.1177/29.4.6166661. [DOI] [PubMed] [Google Scholar]
  11. Korba B. E., Hays J. B. Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA. V. Recombinogenic plasmids from arl mutants of Escherichia coli are unusually sensitive to nuclease S1 and partially deficient in cytosine methylation at C-C-(A/T)-G-G sequences. J Mol Biol. 1982 May 15;157(2):213–235. doi: 10.1016/0022-2836(82)90231-5. [DOI] [PubMed] [Google Scholar]
  12. Liskay R. M., Evans R. J. Inactive X chromosome DNA does not function in DNA-mediated cell transformation for the hypoxanthine phosphoribosyltransferase gene. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4895–4898. doi: 10.1073/pnas.77.8.4895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mohandas T., Sparkes R. S., Shapiro L. J. Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation. Science. 1981 Jan 23;211(4480):393–396. doi: 10.1126/science.6164095. [DOI] [PubMed] [Google Scholar]
  14. Razin A., Riggs A. D. DNA methylation and gene function. Science. 1980 Nov 7;210(4470):604–610. doi: 10.1126/science.6254144. [DOI] [PubMed] [Google Scholar]
  15. Sanger F., Coulson A. R., Friedmann T., Air G. M., Barrell B. G., Brown N. L., Fiddes J. C., Hutchison C. A., 3rd, Slocombe P. M., Smith M. The nucleotide sequence of bacteriophage phiX174. J Mol Biol. 1978 Oct 25;125(2):225–246. doi: 10.1016/0022-2836(78)90346-7. [DOI] [PubMed] [Google Scholar]
  16. Singer J., Roberts-Ems J., Luthardt F. W., Riggs A. D. Methylation of DNA in mouse early embryos, teratocarcinoma cells and adult tissues of mouse and rabbit. Nucleic Acids Res. 1979 Dec 20;7(8):2369–2385. doi: 10.1093/nar/7.8.2369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Singh L., Purdom I. F., Jones K. W. Conserved sex-chromosome-associated nucleotide sequences in eukaryotes. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 2):805–814. doi: 10.1101/sqb.1981.045.01.099. [DOI] [PubMed] [Google Scholar]
  18. Urieli-Shoval S., Gruenbaum Y., Sedat J., Razin A. The absence of detectable methylated bases in Drosophila melanogaster DNA. FEBS Lett. 1982 Sep 6;146(1):148–152. doi: 10.1016/0014-5793(82)80723-0. [DOI] [PubMed] [Google Scholar]

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