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 [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 [3] 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 [4])
  • 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 [5]:
  1. Grow cells to OD600 of ~0.3

References

  1. Bart A, van Passel MW, van Amsterdam K, and van der Ende A. Direct detection of methylation in genomic DNA. Nucleic Acids Res. 2005 Aug 9;33(14):e124. DOI:10.1093/nar/gni121 | PubMed ID:16091626 | HubMed [Bart-2005]
  2. Rao BS and Buckler-White A. Direct visualization of site-specific and strand-specific DNA methylation patterns in automated DNA sequencing data. Nucleic Acids Res. 1998 May 15;26(10):2505-7. PubMed ID:9580708 | HubMed [Rao-1998]
  3. Bach T, Morigen, and Skarstad K. The initiator protein DnaA contributes to keeping new origins inactivated by promoting the presence of hemimethylated DNA. J Mol Biol. 2008 Dec 31;384(5):1076-85. DOI:10.1016/j.jmb.2008.09.042 | PubMed ID:18835566 | HubMed [Bach-2008]
  4. Skarstad K and Løbner-Olesen A. Stable co-existence of separate replicons in Escherichia coli is dependent on once-per-cell-cycle initiation. EMBO J. 2003 Jan 2;22(1):140-50. DOI:10.1093/emboj/cdg003 | PubMed ID:12505992 | HubMed [Skarstad-2003]
  5. Campbell JL and Kleckner N. E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork. Cell. 1990 Sep 7;62(5):967-79. PubMed ID:1697508 | HubMed [Campbell-1990]
All Medline abstracts: PubMed | HubMed

Notes

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