Exon exon junction primers for RT-PCR

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Haritha
Haritha's picture
Exon exon junction primers for RT-PCR

Hi all,

I am having some doubts regarding junction primers.If we design exon exon junction primer, let's say if my gene of interest is having some 5 exons, if the junction primer is designed at 4,5 exons junction, the sequence starting from that junction to rest will be amplified. what about the sequence behind this primer (exon 5). How to get the entire sequence of mRNA in cDNA using junction primers. And also how many junction primers should i design? please kindly clarify my doubts.

Ivan Delgado
Ivan Delgado's picture
Hi Haritha,

Hi Haritha,

Typically the information you get from exon-exon junction primers is to confirm that indeed those are the exon junctions. The rest of the sequence of your cDNA, within the exons, should be available. If it is not, then you can simply run at RT-PCR reaction starting with RNA, clone the cDNA generated and sequence it. That should provide you with all the cDNA sequence of your gene, including the regions between the exon junctions. 

elhamzeinali
elhamzeinali's picture
exon-exon junction primer and its characteristics

I have 2 sets of primer which one primer of each set is designed using exon-exon junction. first set is expected to produce a 117 bp and second one a 184 bp fragment. I have expected PCR product will be produce just with cDNA and for more insurance I used the extracted RNA with these primers. Unfortunately, I got two expected pcr product for cDNA using extract RNA. I have repeated this expriment more and more, but the results was the same. I was really confused and cannot understand what was happened. Is this possible? Can anyone help me with this problem?