Transcription start sites are known, but promoter sequence is not known.
This brings us to the interesting question: How long is a promoter? To my experience, there is no real answer to this question, since it depends on what factors you look.
What do you want to do with the sequence? Use it for activation assays? Do you want to analyse the activation by specific transcription factors? Map sites?
You could at least analyze for transcription factor binding sites and then see how big this piece would be.
I already responded to a similar thread but I didn’t hear back from who post it only if it was what he was looking for. Anyway, I think the best experimental method I know to amplify unknown promoter sequences (or others as well) starting from cDNA or start sites is vectorette PCR. There are kits for it or you can design it yourself. Report back if it is what you are asking.
Activation assays, do not know size of promoter but looking for transcription factor binding sites. what kits do you recommend Abdel and what is vectorette PCR
Then I would run the TF recognition sites through a tool like TESS or RSAT and see where sites are predicted. Then choose the piece according to that information (and also how the sequence looks, not all pieces are easy to amplify, sometimes a slightly larger piece works better). We usually cloned something between 1 and 2 kb.
It might also be useful to do a literature search to see, if somebody already created something like that and try to get it.
I think sometimes we, biologists, are so full of information that we don’t listen to the questions any more. Jeanmarie wants a wet-lab method to PCR-amplify a promoter when only the transcription start sites are known. She doesn’t want at this stage to analyze transcription binding sites which can be done by a combination of in silico approach, functional and biochemical assays.
JeanMarie,
I recommend you get access to Methods in Molecular Biology volume 192 (2002) you can have a more detailed step by step description of the approach as well as other alternatives PCR to amplify unknown neighboring sequences. But in this case because one sequence end is already known, vectorette PCR is the method of choice. Two of my former colleagues in two different labs have done it successfully. Basically there is a digestion, ligation to vectorette units and PCR amplification.
You can buy the Kit from Sigma.
If you don’t have access to MMB, I can send the pdf files to you. Because of the copyright I am not sure I can post it on research gate site.
@Abdelhalim: I can only somehow agree. If you think about cloning a promoter, its important to invest a little time in analyzing it. So you can get to a better result. What is needed for this approach is the information about the genome you are working with. I did my work on promoters in human and mouse and this made things easier.
Of course Christian I understand you and I agree with you, but that is only possible when first you get the promoter sequence. Remember not all genomes have been sequenced. I guess that why JeanMarie was asking an experimental method to amplify a promoter whose sequence is unknown. She only knows the transcription start site probably from a cDNA. She doesn’t yet know what is the sequence of the promoter
There are specific promoter elements that I can examine and I appreciate the recommended program. I was considering TAIL_PCR but was wondering if there were additional options without building a library. Vectorette is one I had not considered. That looks like a library or BAC based approach?
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