JBiSE  Vol.3 No.5 , May 2010
A stage-scanning laser confocal microscope and protocol for DNA methylation sequencing
ABSTRACT
Recent understanding of the role of epigenetic regulation in health and disease has necessitated the development of newer and efficient methods to map the methylation pattern of target gene. In this article we report construction of a stage-scanning laser confocal microscope (SLCM) and associated protocol that determines the methylation status of target gene. We have adapted restricted Sanger’s sequencing where fluorescine labeled primers and dideoxy guanine fraction alone are used for target amplification and termination at cytosine positions. Amplified ssDNA bands are separated in 6% denaturing PAGE and scanned using SLCM to sequence the positions of methylated cytosines. We demonstrate that our me- thodology can detect < 100 femtomoles of DNA, and resolve the position of cytosine within ± 2 nucleotide. In a calibration run using a designer DNA of 99 bases, our methodology had resolved all the 11 cytosine positions of the DNA. We have further demonstrated the utility of apparatus by mapping methylation status in the Exon-1 region of a gene, E-Cadherin, in the plasma DNA sample of a healthy subject. We believe our approach constitute a low cost alternative to conventional DNA sequencers and can help develop methylation based DNA biomarkers for the diagnosis of disease and in therapeutics.

Cite this paper
nullVaishnavi, V. , Varghese, L. and Ali, B. (2010) A stage-scanning laser confocal microscope and protocol for DNA methylation sequencing. Journal of Biomedical Science and Engineering, 3, 496-500. doi: 10.4236/jbise.2010.35069.
References
[1]   Li, E., Bestor, T.H. and Jaenisch, R. (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell, 69(6), 915-926.

[2]   Jones, P.A. and Baylin, S.B. (2007) The epigenomics of cancer. Cell, 128(4), 683-692.

[3]   Toyooka, S. and Shimizu, N. (2004) Models for studying DNA methylation in human cancer: A review of current status. Cancer, 1(1), 37-42.

[4]   Post, W.S., et al. (1999) Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system. Cardiovascular Research, 43(4), 985-991.

[5]   Anselmo, N.P., et al. (2006) Epigenetic alterations in human brain tumors in a brazilian population. Genetics and Molecular Biology, 29(3), 413-422.

[6]   Laird, P.W. (2003) The power and the promise of DNA methylation markers. Nature Reviews Cancer, 3(6), 253- 266.

[7]   Eick, D., Fritz, H.J. and Doer, W. (1983) Quantitative determination of 5-methylcytosine in DNA by reverse- phase high-performance liquid chromatography. Analytical Biochemistry, 135(1), 165-171.

[8]   Fraga, M.F. and Esteller, M. (2002) DNA methylation: A profile of methods and applications. BioTechniques, 33(3), 632-649.

[9]   Jean-Michel, D., Jorg, T., Helene, J. and Ivo, G.G. (2004) De novo quantitative bisulfite sequencing using the pyrosequencing technology. Analytical Biochemistry, 333 (1), 119-127.

[10]   Grigg, G.W. and Clark, S.J. (1994) Sequencing 5-methy- lcytosine residues in genomic DNA. Bioessays, 16(6), 431-436.

[11]   Herman, J.G., Graff, J.R., Myohanen, S., Nelkin, B.D. and Baylin, S.B. (1996) Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proceedings of the National Academy of Sciences, 93(18), 9821-9826.

[12]   Stach, D., et al. (2003) Capillary electrophoretic analysis of genomic DNA methylation levels. Nucleic Acids Research, 31(2), e2.

[13]   Michaela, W.D.S., Hans-Christian, K., Manfred, W. and Oliverl J.S. (2004) Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser-induced fluorescence detection. Electrophoresis, 25(6), 839-845.

[14]   Clark, S.J., Harrison, J., Paul, C.L. and Frommer, M. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Research, 22(15), 2990-2997.

[15]   Warnecke, P.M., Stirzaker, C., Melki, J.R., Millar, D.S., Paul, C.L. and Clark, S.J. (1997) Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Research, 25(21), 4422-4426.

[16]   Sambrook, J. and Russell, D.W. (2000) Molecular cloning A laboratory manual. 3rd Edition, Chapter 6.

[17]   Marianne, F., et al. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proceedings of National Academy of Sciences, 89(5), 1827-1832.

[18]   http://www.urogene.org/methprimer/index1.html

[19]   Vaishnavi, V., Aarthi, R., Smitha, S. and Jaffar Ali, B.M. (2010) A simplified method to determine methylated cytosines in a target gene. IEEE Explore Conference Proceedings, in Press.

[20]   Victoria, L.B., Kristina, I.M., Achim, E.K., Kenneth, J.L. G.Z. and John, W.B., (2006) Methylation-dependent frag- ment separation: Direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite- converted genomic DNA. Analytical Biochemistry, 354(2), 266-273.

 
 
Top