Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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DNA Bisulfite Sequencing for Single-Nucleotide-Resolution DNA Methylation Detection

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DNA methylation plays an important role in multiple biological processes. Therefore, methodologies that can detect changes in DNA methylation are of general importance. Currently, the most popular and reliable method for measuring DNA methylation status is DNA bisulfite sequencing (Clark et al. 1994). Denatured DNA (i.e., single-stranded DNA) is treated with sodium bisulfite under conditions that preferentially convert unmethylated cytosine (C) residues to uracil (U) residues while methylated cytosines remain unmodified (Fig. 1). The converted DNA can then be amplified using a gene-specific primer. U is amplified as thymidine (T) in PCR and detected as T during DNA sequencing. Note that bisulfite conversion does not discriminate between 5-methylcytosine and 5-hydroxymethylcytosine (Fig. 1). 5-Hydroxymethylcytosine is a newly identified cytosine modification observed in embryonic stem cells and Purkinje neurons (Kriaucionis and Heintz 2009; Tahiliani et al. 2009).


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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