c-Fos Methylation

DNA Methylation sites in c-Fos promoter

Normal Rat Liver, From Shimizu et al., Cancer Sci 2007; 98: 1318–1322

Approximate MW:

38,337 daltons (unmethylated)

38,252 daltons (methylated)

each methylation site causes 15 dalton decrease in MW 

Rat c-fos Primers used by Alcala and Houpt

Primers designed with Primer 3 using the 300 bases upstream of c-Fos transcription start based on gi|62750806|ref|NC_005105.2|NC_005105 (equivalent to bases 172-471 of U02631, which claims start of 5’UTR is at 455.)

Forward at -261 (cFosSense-261):

5'-CCATCCCCGAAATCCTACAT-3'

Reverse at -6 (cFosAS-6):

5'-CAATCGCGGTTGGAGTAGTA-3'

Product size: 256 bp

PCR conditions for use with HotStarTaq mix (progam CFOSGENO on thermocycler)

95° for 15 min to activate Taq

Repeat 35 times:

94° for 1 min

59° for 1 min

72° for 1 min

72° for 10 min final extension

4° for overnight

Sequenced PCR product from Brain and Tail amplification on 1/29/09

Matched AY786174.1 base pairs 85-339

ccatcc ccgaaatcct acatgcggag ggtccaggag accttctaag atcccaattg tgaacactca taggtgaaag
ttacagactg agacgggggt tgagagcctg gggcgtagag ttgatgacag ggagcccgca gagggcattc
gggagcgctt  tcccccctcc agtttctctg ttccgctcat gacgtagtaa gccattcaag cgcttctata aagcggccag
ctgaggcgcc tactactcca accgcgatt

Rat c-fos Primers from Literature

From Shimizu et al., Cancer Sci 2007; 98: 1318–1322

Bisulfite sequencing (Genomic DNA)

GenBank AF126534

F: 5′-TATTTATAGGTGAAAGTTATAGATTG-3′

R: 5′-CACTAATAAAAACTACAAAACAAAACT-3′

Annealing temperature 54

Product size 292 bases

4-23-08 -- could NOT confirm with blast -- need to check earlier references

RT-PCR (from RNA)

GenBank X06769

F: 5′-TTGCGCAGATCTGTCCGTCT-3′

R: 5′-GTTGATCTGTCTCCGCTTGG-3′

Annealing temperature 65

4-23-08 -- confirmed with blast