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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.

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• TITLE: A stage-scanning laser confocal microscope and protocol for DNA methylation sequencing

  AUTHORS: Vaithilingam Vaishnavi, Litty Varghese, Baquir Mohammed Jaffar Ali

  KEYWORDS: Scanning Confocal Microscope; Fluorescence Detection; Methylation; Bisulfite Sequencing; DNA Sequencer

  JOURNAL NAME: Journal of Biomedical Science and Engineering, Vol.3 No.5, May 27, 2010

  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