User:Torsten Waldminghaus/Notebook/Quantification of Methylation by Sequencing
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It was found that methylated nucleotides change incorporation of modified nucleotides used for DNA sequencing [1, 2]. This makes it possible to destinguish between methylated and unmethylated sites in DNA by sequencing. The goal of this project is to find if it is also possible to quantify this to find out how much of the DNA is methylated. This is a common question usually answered with Southern Blot experiments which are quite workintensive. To see if there is a correlation of peak intensity with the degree of methylation the first step is to sequence defined mixtures of plasmids from an E. coli dam- strain with plasmids from a dam+ strain that were also methylated in vitro. For the first experiments I used plasmid pGATC  that containes a GATC cluster with numerous methylation sites. Complete in vitro methylation of the dam+ pGATC was checked by digestion with HphI which is sensitive to methylation (2.10.2008).
Strains and Plasmids
Method for Plasmid Isolation