User:Torsten Waldminghaus/Notebook/Quantification of Methylation by Sequencing

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Project Description/Abstract
It was found that methylated nucleotides change incorporation of modified nucleotides used for DNA sequencing Bart-2005; Rao-1998. 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 Bach-2008 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

 * MOR2		ALO14(delta oriC1071, hfr::ilv, asnA::Tn10,asnB) lacZ:Tn5		km, tet
 * ALO14	lacZ:Tn5 tet	Box:O21
 * ALO1826	ALO14/pOC24 #1 Ref. MGG162, 269-275 (1978)		amp		Box:R41
 * ALO1829	ALO1826dam13::Tn9. Constr ALO1826XP1(ALO348) Sel	amp, cam	Box:R43

Future Plans

 * Use the method to analyse methylation of oriC on the minichromosome (plasmid with chromosomal origin):
 * 1) Purify minichromosome (pOC24) from dam+ (ALO1826) and dam- (ALO1829) strains (see strainlist above and Skarstad-2003 )


 * methylation in different strains could be interesting:
 * Dam-overproduction
 * seqA deletion, over-production, under-production
 * synchronized cells
 * DnaA-over and underproduction (deletion)

Method for Plasmid Isolation

 * When isolating plasmids that should be analysed regarding there methylation state it is important to stopp Dam methylase as fast as possible after taking samples. The following method is adapted from Campbell-1990 :
 * 1) Grow cells to OD600 of ~0.3