primers for promoter amplification - (Apr/09/2007 )
I'm about to do an experimental promoter analysis, to do that I've already been running some bioinformatic analysis with Genomatix tools....
Based on the results I have a predicted promoter sequence of 617bp that starts 516bp Upstream of the first exon, then it includes the 81bp exon and it ends 20bp downstream from the exon. Is it OK? and to start with experimental analysis should I amplified the whole sequence from the genome? (exon included).
The exon is not translated, is that important?
As for the TSS, it is predicted at position 501.
By the way, do you have a good protocol for genomic DNA purification from cell lines (human)?
why not use more sequence upstream sure most promoters reside within 500bp but why bet on that, use about 2kb upstream and start 5' end deletions from there. you can use the downstream sequence also, in some cases that has been shown to be important, the problem may come in with shifting the translation frame etc and artificially impacting the reporter, so I stopped at or near the transcription start site to avoid that issue. No it does not matter that the sequence is not translated this is where transcription begins and that is what you care about. There is an ATG in the construct for translation of the reporter gene... again watch out for effects that are not really due to promoter activity ie your UTR exon inhibits translation etc... I suggest TriReagent or Trizol for DNA isolation. BTW always smart to experimentally verify the TSS if it has not already been done...