not too sure why you chose 340bp upstream of the gene.

I routinely use Genome Browser, it's easier to navigate the database than NCBI and ensembl is some ways.

I have attached the grapical readout of this gene, there is indeed a CpG islands at the 5' end.

As for the differences between ensembl and NCBI, I am not too sure about that one.

Good luck

nick

Hi,I am very glad to see posts about methylation protocol here.
I have already tried in this aspect for a long time.I bought a kit about p16 ,modification and methylation.And I tried with Human samples.I have gotten very good results,and the bands were very clear.Then I tried with mouse samples,extracted DNA from mouse spleen,modificated and amplified with the primer designed like pcrman mentioned.But no bands appeared.And I tried with more primers like p15,pten,etc,still no band.
Could you please tell me what is my problem?
Comparing with the kit for human(no kit for mouse),there is only difference with primers.I wonder if I should modificate the primer designed with methprimer?
I am a new postdoc here and I just can not do this well.I am very hopeful for your help.Thanks!

Hi Guys,

Quick question for you all, and if this is a really simple molecular queston I apologize. So I'm working on designing primers for my gene of interest and have found a CpG island immediately upstream from the 5' exon. However, searching through this section of sequence I cannot find the TATAA box or the TFIID/TBP binding locations. (using TESS)

How do you all normally go about determining where exactly in your promotor the start site is located? The CpG island i suspect includes the promotor region extends into the first exon as well. Is it even possible for the transcriptional start site to be found within an exon?

Somewhat confused. Any help would be appreciated

Thanks

Hi pcrman, I'm trying to study a gene promoter too,

I've already been analyzing with several programs a region upstream of the first exon (I analyzed and compared the results of 500bp, 1000bp, 2000bp and 4000bp upstream the exon 1) but I had different results with each program, what should I do?
First of all, why do we first do a bioinformatic analysis of this region? and don't start the experiments with a reporter gene directly?
how do I do to know if my promoter has CpG islands, and......what is the methylation mapping used for?.................and what do I do if the promoter doesn't have islands?
I've been trying to used the methprimer and I had a lot of predicted CpG islands; I just have to see if there is one near the first exon? and how can I see the information in a graphic like the one you show in the topic?

As you might see I'm not very smart at this

Hope you can help me...

Bio...

Hi Biotech!

Methprimer produces the graphic output PCRman has posted. The bioinformatics approach is chosen as itdoes not always make sense to work with reporter genes when all you are interested in is, if differences in DNA methylation occur + you need this approach for sufficient primer design. The differences for CpG-island prediction in different programs can be explained by different algorithms for CpG island prediction. ABI's MethylPrimerExpress software lets you change almost all of these conditions to suit your needs.
For your gene: it often makes sense to start with a CpG island around the transcritption start site of exon 1, for other genes ist can also be worth looking at the ATG ... There are no general rules on that. It can be helpful to use Mathinspector to look, if there are interesting TF binding sites within your CpG island (for example CREB often binds to sites within CpG islands).
If the promoter doesn't contain CpG islands, methylation is not so likely to be of great relevance...

Hope this helps you ...

Krümel

When researching CpG islands in promoters, I often found that the CpG island immediately upstream of exon1 reached quite far into the exon. Sometimes even the majority of the island's CpGs were actually part of the exon, and not upstream of it.
How important are those exonic CpGs for the promoter or, more directly: the transcription of the gene? Would you include them in your BSP? Are they likely to influence promoter binding and HDAC recruitment / histone modification etc?

Short answer: Yes. CpG islands often span promoter and exon 1. The exonic methylation seems to be as relevant as the promoter methylation. There are also reports on relevant methylation in downstream exons and introns (there are some threats about that in the forum). If nothing is known about the methylation of your gene of interest it seems to be a good idea to look at the whole promoter/exon 1 CpG island ... or guess a position and hope that it is related to the expression

HI,I'M NEW HERE...I WILL WORK WITH p53 HUMAN GENE...I HAVE TRIED A LOT TO FIND ARTICLES ABOUT HOW TO DESIGN PRIMERS FOR MSP...ALL OF THESE SUGGEST TO USE SEVERAL ALGORYTHMS...BUT WICH IS THE PROCESS WHEN SOMEONE WANT TO DESIGN MSP-PRIMERS??THANKS FOR ANY HELP...I"M DESPERATE!