From the Microarray data (based on the EST clone), I found several genes that needs deeper research to find out the gene expression under heavy metal stress. Thus, we need to conduct an qPCR to analyze the gene expression, but is it possible to construct a PCR primer based on the Microarray data/EST Clone which just a short sub sequence from the cDNA? Since it's full sequence is not yet known, that's also another problem (I'm researching about A. halleri mechanism under heavy metal stress).
And also, if I want to make a PCR primer for normal PCR (from DNA and not cDNA) is it possible? Because PCR Primer for PCR and qPCR is different since qPCR template is from cDNA.
Thank you for your help!
Hi Christine,
You could try to design qPCR primers based on the ESTs. It could be enough to have 200 bp if there is a good region for qPCR within this short sequence. Do you have information abut the exon intron structure of your gene? qPCR primers should span exon exon borders to prevent the amplification of genomic DNA.
Have you looked for your gene of interest in the available sequences of close relatives like A. thaliana and A.lyrata? I know that it is unlikely to find all genes, but you could get important information if some of your genes are present in these sequences.
The PCR on genomic DNA would be easier with information from the relatives e.g. expected size of the PCR product. However, primers designed on cDNA should also work on gDNA. To avoid primers across exon exon borders you could design two primer pairs by taking the sequence upstream or downstream of your initial primers.
Good luck!
Hi Boas Pucker,
Thank you so much for your answer, Sadly I've searched for the exon intron structure of the gene sequence but I can't find it for A. halleri, There're similar gene sequence in A. thaliana, however, I'm not sure I can use the same primer to amplify similar genes in A. thaliana and A. halleri.
So it is possible to use the same primer for gDNA using primer from cDNA? Because what I'm afraid of is because the cDNA only contain exon and gDNA contain both so there's a possibility that the primer didn't match.
Thanks again for your help!
Hi Christine,
The cDNA primers should match the gDNA as well. The risk of designing a primer across an exon exon border exists, but is relatively small. However, you cannot amplify the entire sequence of your gene of interest if you rely on the EST information.
The A. halleri genome should be already sequenced. Have a look at phytozome:
https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Ahalleri_er
Best,
Boas
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