Exome data obtained from NGS analysis show the mutation in PMS_2 gene. I would like to confirm the mutation by Sanger sequencing but the PMS2 have a Pseudogene PMS2CL. I'm trying to isolate the sequence by LR-PCR and then nested PCR but I can not confirm the mutation because I supposed that my primers annealing with the sequence of PMS2 pseudogene.
if the pseudogene is not expressed can you get cdna from a sample and sequence that?
alternatively primers in the introns either side of your mutated exon could be designed within sequences which exist only in the gene sequence .Even a one base change at the 3'end of the primer sequence is often enough to stop one sequence amplifying but allow another sequence to amplify
thank you. I' ll follow this method. It s a goog idea sequencing cDNA! I thought this but I supposed it is not possible to do it.
You can also digest your gDNA sample with a restriction enzyme that cuts the pseudogene seq but not the gene. You can then amplify with your primers as the restricted pseudogene sequence will not give a PCR product.
The candidate gene is PMS2. The sample which i ll extract RNA is blood. I don t know if the RNA is circulating. But I m finding this....thank you to all.
ideally amplify from adjacent exons just in case the is some genomic dna contamination present . If there is very little cdna you can always use nested pcr with internal primers to get great sensitivity of amplification
Can't you just use a duplex taqman assay with one probe for you WT and another probe for your mutation?
I expect that you will find some in blood. Often cells which are stressed will express unusually and there is a good deal of white cell stress in the process of bleeding, storage in a blood tube and DNA/rNA production
Best approach is to isolate RNA and work from cDNA. Exon 10 (I believe) is unique to the "real" PMS2 gene so try to do a (longe range?) first PCR using one primer in exon 10, followed by a nested PCR. There are quite some papers (vd Klift et al) that describe this procedure.
Dear Mark,t he mutation is in the exsons 2 that have high omology with 5' region of PMS2Cl(pseudogene). I already tried with LR-PCR and subsequently Nest PCR for exson 2 but I think that Primers anniling with Pseudogene.
You can use gDNA and choose one longrange PCR primer in the unique exon 6, or you can use cDNA but you should use RNA from shortcultured lymphocytes or fibroblasts in which you can inhibit nonsense mediated decay. For PCR of cDNA you should also use an amplicon with at least one primer in a unique PMS2 exon (exons 6-8 and 10).
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