User:Torsten Waldminghaus/Notebook/Methylation array: Difference between revisions
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===MseI Restriction=== | ===MseI Restriction=== | ||
*Digest 500ng chrom. DNA with 10U of MseI in 10μL volume for 3h. | |||
*Heat inactivate for 20 min at 65°C | |||
===Linker Ligation=== | ===Linker Ligation=== | ||
* | *Annealing of oligos MseILong (5' AGTGGGATTCCGCATGCTAGT 3') and MseIShort (5' TAACTAGCATCG 3')<cite>Watson-2004</cite>: | ||
**Mix 1μL of 100μM (=100pmol/μL) stocks of both primers with 4μL H<sub>2</sub>O in PCR tube | |||
**Run program starting at 75°C and cooling down to 15°C with 1°C per 2 minutes. | |||
*At 15°C add 10μL MseI cut DNA, 2μL Ligase Buffer and 2μL Ligase | |||
*Ligate over night | |||
==One elongation round== | |||
<biblio> | <biblio> |
Revision as of 04:54, 15 July 2008
Idea
- Analyse the methylation of GATC sites genomewide in E. coli.
Notes
- Løbner-Olesen et al, 2003 [1] used a aroK17::cat strain to have more hemimethylation in the cell, since a polar effect on dam leads to a reduced Dam content in the cell (only 30% of wt [2]). Could be used as control and for interesting analysis.
- methylation in different strains could be interesting:
- dam-overproduction
- seqA deletion, over-production, under-production
- synchronized cells
- does introduction of a GATC cluster alter methylation of surrounding sites?
- what about GATC sites in datA? sequestration?
- one could compare methylation in protein coding regions with intergenic regions and RNA coding.
- one could analyse effect of distance to oriC and density of GATCs
- REN to cut the chrom. DNA in the first step could be MseI which was used before [3]. It cuts TTAA and can be heat inactivated at 65 °C for 20 min (http://www.neb.com/nebecomm/products/productR0525.asp).
- As independent method and first step one could analyse cutting by methylation sensitive REN with qPCR
- on whole chromosome
- clusters and isolated GATCs
- coding regions and intergenic regions
Possible restriction enzymes that are Dam methylation sensitive:
Enzyme | Site | Notes | Link |
---|---|---|---|
HphI | GGTGA | No star activity; reaction at 37°C; heatinactivation at 20 min 65°C | http://www.fermentas.com/catalog/re/hphi.htm |
MboII | GAAGA | Star activity; reaction at 37°C; heatinactivation at 20 min 65°C | http://www.fermentas.com/catalog/re/mboii.htm |
TaqI | TCGA | No star activity; reaction at 65 °C; no heatinactivation after 20 min 80°C | http://www.fermentas.com/catalog/re/taqi.htm |
- HphI seems to be a goog choice since it can be heatinactivated and has no star activity (does not cleave unspecific when DNA is overdigested).
- In unsynchronized cultures the detection of hemimethylation will be difficult. If every GATC is hemimethylated for about 1 min and replication of the chromosome takes about 50 min, than about 2% of a specific GATC will be in a hemimethylated state giving only 1% cut with an overlapping enzyme.
- One possibility to make the restriction outcome higher could be to use sites like ggtgatcacc that have two HphI restriction sites overlapping one GATC. This should give a doubling in cutting at that site compared to only one HphI site. However, a 10bp long inverted repeat might not give a representative result for GATC methylation because some whatever protein could bind there. The E. coli K12 genome contains 33 ggtgatcacc sites:
Start End Pattern_name Mismatch Sequence 118460 118469 pattern1 . GGTGATCACC 143911 143920 pattern1 . GGTGATCACC 210581 210590 pattern1 . GGTGATCACC 282472 282481 pattern1 . GGTGATCACC 330621 330630 pattern1 . GGTGATCACC 379605 379614 pattern1 . GGTGATCACC 405197 405206 pattern1 . GGTGATCACC 599247 599256 pattern1 . GGTGATCACC 636380 636389 pattern1 . GGTGATCACC 681039 681048 pattern1 . GGTGATCACC 685084 685093 pattern1 . GGTGATCACC 712647 712656 pattern1 . GGTGATCACC 826095 826104 pattern1 . GGTGATCACC 834158 834167 pattern1 . GGTGATCACC 854836 854845 pattern1 . GGTGATCACC 900826 900835 pattern1 . GGTGATCACC 1065260 1065269 pattern1 . GGTGATCACC 1107328 1107337 pattern1 . GGTGATCACC 1363023 1363032 pattern1 . GGTGATCACC 2152717 2152726 pattern1 . GGTGATCACC 2358426 2358435 pattern1 . GGTGATCACC 2540379 2540388 pattern1 . GGTGATCACC 2953039 2953048 pattern1 . GGTGATCACC 3010031 3010040 pattern1 . GGTGATCACC 3042067 3042076 pattern1 . GGTGATCACC 3268923 3268932 pattern1 . GGTGATCACC 3528134 3528143 pattern1 . GGTGATCACC 3540084 3540093 pattern1 . GGTGATCACC 3607305 3607314 pattern1 . GGTGATCACC 3858080 3858089 pattern1 . GGTGATCACC 4238710 4238719 pattern1 . GGTGATCACC 4292770 4292779 pattern1 . GGTGATCACC 4463247 4463256 pattern1 . GGTGATCACC
Name (localisation of HphI site) | feature in this region | MseI sites | GATCs | Sequence | Size of MseI fragment | Oligos for qPCR | |
---|---|---|---|---|---|---|---|
761873 | sucB CDS | 761873_P CGAATCCGTGGGCTTCCTGG 761873_fw GAGATCCTGCCGATGATGTA 761873_rv TTCCAGCAACTCTTTGATCG |
- One other possibility to check the hemimethylation in general is to isolate chrom. DNA and than methylate it with radioactive SAM. The amount of incorparated radioactivity should be proportianal to the hemi or unmethylated DNA. As control one could use the same DNA that was incubated with Dam and non radioactive SAM before. Defined substrates as for example a plasmid from dam- strain could help to normalize the found radioactivity and calculate how many GATCs were hemimethylated.
Protocol REN-qPCR
Isolate DNA from MG1655
- grow cells
- take 2x 10ml samples at OD 0.15 and transfer directly to 10 ml TE with 3% SDS at 65 °C (7ml TE + 3ml 10% SDS)
- leave at 65 °C for 5 min
- add 10 ml Isopropanol and store at -80 °C for 30 min
- centrifuge hith speed for 20 min
- wash in 70% ethanol and transfer to 2 ml reaction tubes
- resuspend in 200 μL A. dest
- add 1 μL RNase A (27.5 mg/ml) and incubate 30 min at 65 °C
- add 10 μL proteinase K (20 mg/ml) 1h at 50 °C (endconcentration = 1 mg/ml).
- extract with phenol/chlorophorm
- preciptitate with ehanol and Na-acetate
- resuspend in 50 μL A. dest and meassure at Nanodrop
Protocol Methylation Array
DNA isolation
MseI Restriction
- Digest 500ng chrom. DNA with 10U of MseI in 10μL volume for 3h.
- Heat inactivate for 20 min at 65°C
Linker Ligation
- Annealing of oligos MseILong (5' AGTGGGATTCCGCATGCTAGT 3') and MseIShort (5' TAACTAGCATCG 3')[4]:
- Mix 1μL of 100μM (=100pmol/μL) stocks of both primers with 4μL H2O in PCR tube
- Run program starting at 75°C and cooling down to 15°C with 1°C per 2 minutes.
- At 15°C add 10μL MseI cut DNA, 2μL Ligase Buffer and 2μL Ligase
- Ligate over night
One elongation round
- Løbner-Olesen A, Marinus MG, and Hansen FG. Role of SeqA and Dam in Escherichia coli gene expression: a global/microarray analysis. Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4672-7. DOI:10.1073/pnas.0538053100 |
- Løbner-Olesen A, Boye E, and Marinus MG. Expression of the Escherichia coli dam gene. Mol Microbiol. 1992 Jul;6(13):1841-51. DOI:10.1111/j.1365-2958.1992.tb01356.x |
- Pfister S, Schlaeger C, Mendrzyk F, Wittmann A, Benner A, Kulozik A, Scheurlen W, Radlwimmer B, and Lichter P. Array-based profiling of reference-independent methylation status (aPRIMES) identifies frequent promoter methylation and consecutive downregulation of ZIC2 in pediatric medulloblastoma. Nucleic Acids Res. 2007;35(7):e51. DOI:10.1093/nar/gkm094 |
- Watson SK, deLeeuw RJ, Ishkanian AS, Malloff CA, and Lam WL. Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays. BMC Genomics. 2004 Jan 14;5(1):6. DOI:10.1186/1471-2164-5-6 |
- Braun RE and Wright A. DNA methylation differentially enhances the expression of one of the two E. coli dnaA promoters in vivo and in vitro. Mol Gen Genet. 1986 Feb;202(2):246-50. DOI:10.1007/BF00331644 |
- Kücherer C, Lother H, Kölling R, Schauzu MA, and Messer W. Regulation of transcription of the chromosomal dnaA gene of Escherichia coli. Mol Gen Genet. 1986 Oct;205(1):115-21. DOI:10.1007/BF02428040 |
- 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. DOI:10.1016/0092-8674(90)90271-f |
- Riber L, Olsson JA, Jensen RB, Skovgaard O, Dasgupta S, Marinus MG, and Løbner-Olesen A. Hda-mediated inactivation of the DnaA protein and dnaA gene autoregulation act in concert to ensure homeostatic maintenance of the Escherichia coli chromosome. Genes Dev. 2006 Aug 1;20(15):2121-34. DOI:10.1101/gad.379506 |
- 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 |
- 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. DOI:10.1093/nar/26.10.2505 |