I want to amplify the promoter region for one cloned gene in rice. For this I'm going to amplify 2 kb region (from genomic DNA) before the start codon (ATG). I have a concern regarding the reverse primer that how much freedom we have to choose the better reverse primer before ATG start codon. And also in relation to 5'UTR.
2 kb is a rule of thumb in many plants. Promoters could always be complicated. Check the annotated neighboring genes. If there aren't any with in 2 kb and no info on any important regions then 2 kb upstream of the start codon is the way. What do you try to do (your objective of cloning)?
Shajahan
If your gene is annotated, then you should be able to get a good estimate of the promotor location using bioinformatics tools
Hi Atul,
It depends on the application. Generally speaking the reverse primer needs to be positioned right beside the translation initiation site to ensure that the minimal promoter (TATA Box and Y patch) is included. However if you are providing a different minimal promoter, for example the minimal promoter from CamV35s, then the reverse primer can be located further upstream than the start codon.
Use EPD [Eukaryotic Promoter Database]. You can find upstream elements easily. I have attached the link below.
http://epd.vital-it.ch/cgi-bin/get_doc?db=mmEpdNew&format=genome&entry=Wap_1
Hi atul.....
You can do primer walk sequencing upto 3 kb or more with primer designed at 5' (nearer to atg seq)end, using genomic DNA as template. once you get the contig you can predict the promoter sequence using promoter prediction tool. But once you get the promoter sequence you need to validate this promoter by cloning and expression.
Thank you Sachin Sir.
@Shajahan, I wish to know in which tissue/plant part our candidate gene expressing.
@Ben Paters, I want to clone the native promoter of our candidate gene in the vector containing GUS gene. Thank you Chanuka.
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