There are three CpG sites (cg02228185 in ASPA, cg25809905 in ITGA2B, and cg17861230 in PDE4C). I would like to find sequences of them.
I have googled and gone to ncbi, but am still clueless..
Bisulfite specific primers for these three sites are included in these supplemental materials:
https://static-content.springer.com/esm/art%3A10.1186%2Fgb-2014-15-2-r24/MediaObjects/13059_2013_3218_MOESM1_ESM.pdf
to this paper:
http://genomebiology.biomedcentral.com/articles/10.1186/gb-2014-15-2-r24#CR41
The primers can be used for bisulfite specific in silico PCR at this site:
http://bisearch.enzim.hu/?m=genompsearch
For the ASPA primers, the result returned is this:
PCR product(s) on the bisulfite transformed sense chain
Forward primer: ATTATTTGGTGAAATGATT
Reverse primer: CAACCCTATTCTCTAAATCTC
1. Chromosome 17 (len: 151)
3476146 ATTATTTGG TGAAATGATT TTTTGTTATA TTGTTGAAGA ATATATATAA
AAGGTTGTTA TTTTTGGAGG AATTTATGGG AATGAGTTAA TTGGAGTATT
TTTGGTTAAG TATTGGTTAG AGAATGGTGT TGAGATTTAG AGAATAGGGT
TG 3476297
The coordinates shown are for genome build:Homo sapiens GRch 38.83.
You can obtain the "normal" genomic sequence by going to the genome browser gateway,
http://genome.ucsc.edu/cgi-bin/hgGateway
Select human genome build 38 and entering the coordinates shown in the format chr71:3,476,146-3,476,297 into the search box.....returning:
http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg38&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=chr17%3A3476146-3476297&hgsid=480626633_SpZtxmzaIS5afMfhpVpjI6GcZ2W7
They are CpG IDs from Illumina's methylation array. You should be able to find the position and sequence on Illumina's website.
Bisulfite specific primers for these three sites are included in these supplemental materials:
https://static-content.springer.com/esm/art%3A10.1186%2Fgb-2014-15-2-r24/MediaObjects/13059_2013_3218_MOESM1_ESM.pdf
to this paper:
http://genomebiology.biomedcentral.com/articles/10.1186/gb-2014-15-2-r24#CR41
The primers can be used for bisulfite specific in silico PCR at this site:
http://bisearch.enzim.hu/?m=genompsearch
For the ASPA primers, the result returned is this:
PCR product(s) on the bisulfite transformed sense chain
Forward primer: ATTATTTGGTGAAATGATT
Reverse primer: CAACCCTATTCTCTAAATCTC
1. Chromosome 17 (len: 151)
3476146 ATTATTTGG TGAAATGATT TTTTGTTATA TTGTTGAAGA ATATATATAA
AAGGTTGTTA TTTTTGGAGG AATTTATGGG AATGAGTTAA TTGGAGTATT
TTTGGTTAAG TATTGGTTAG AGAATGGTGT TGAGATTTAG AGAATAGGGT
TG 3476297
The coordinates shown are for genome build:Homo sapiens GRch 38.83.
You can obtain the "normal" genomic sequence by going to the genome browser gateway,
http://genome.ucsc.edu/cgi-bin/hgGateway
Select human genome build 38 and entering the coordinates shown in the format chr71:3,476,146-3,476,297 into the search box.....returning:
http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg38&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=chr17%3A3476146-3476297&hgsid=480626633_SpZtxmzaIS5afMfhpVpjI6GcZ2W7
Thank you for the answer...
What a relief after having got the answer so easily...
It was Too hard for me to work on it alone. Really appreciate this.
When I blasted the primer sequences in the paper in ncbi, I could not find ASPA as the result.
I guess here the primers primers for the sequence of ASPA gene after bisulfite treatment. Am I right on this part? If that is not the reason, what is the reason for not being able to detect ASPA after blast?
One more thing is this site http://bisearch.enzim.hu/?m=genompsearch is not working for PDE4C gene...do you know the reason for this ??
Anyways, I am very very very very grateful for your help. Really very very very.
@Chanwoo:
I guess here the primers primers for the sequence of ASPA gene after bisulfite treatment. Am I right on this part?
YES!
One more thing is this site http://bisearch.enzim.hu/?m=genompsearch is not working for PDE4C gene...do you know the reason for this ??
This primer pair does work. (My earlier version of this post was in error!)
PCR product(s) on the bisulfite transformed antisense chain
Forward primer: AGGTTTGTAGTAGGTTGA
Reverse primer: AACTCAAATCCCTCTC
1. Chromosome 19 (len: 156)
18233004 AACTCA AATCCCTCTC
ACAACAACCC ACAAACTTAT CCAAATCCAA ATAAAAACAC TATTAAATAC
AAATAAAACA CCAAAATTAC CACCACAAAT ACTTCAAAAC TCTAATCATA
CTATAACAAC CACAAAAACA ACTCAACCTA CTACAAACCT 18233160
And these coordinates do correspond to the PDE4C promoter/CpG island.
http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg38&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=chr19%3A18233004-18233160&hgsid=480699137_esrBX1KM7kB5jytAq8drCzx5FAcN
Regarding PDE4C: published primer sequence of reverse PCR primer seems to be correct, doesn't understand Davids remark on 3'C??
5 ́-AACTCAAATCCCTCTC-3 ́, that's a well working reverse primer for above PCR of PDE4C.
@Uwe. Thanks for pointing that out! I had a cut and paste mishap! I've corrected my earlier post.
I thing the question was how does one locate the sequence cg02228185 in ASPA. This CpG site not in the gene itself which is on human chromosome 17. Perhaps this is corresponding to an upstream promoter region or cis acting elelment?
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