Greetings everyone, I was wondering if I could run something by you all?
I have been running gels to confirm PCR product size of primers. I have 6 primer pairs to design for 6 genes, and so far I have confirmed standard curve efficiency and product sizes for 4 of these genes.
However, the final two genes are proving to be very challenging. In summary, I have designed 3 primer pairs per gene with the idea that I choose the pairs that return the best efficiency, and then confirm product sizes on a gel. I have ran standard curves ( 1 in 2 serial dilution) for the primers and I had some primer pairs return good efficiency values, R values and error. However, I have ran these products on a gel (the least diluted PCR products) and the product sizes are much higher than they should be, some by over 100bp's and one by up to 400 bp's. I thought at first this might be because I had the elongation step at 24 seconds during the qPCR, but I have reduced the elongation to the standard 20 seconds and nothing has changed. An elongation step of 20s has worked for all of my other genes, even for pairs that have a product size of 140 bp's.
I also thought that this is potentially due to gDNA contamination, yet the RNA was Dnase-treated during extraction and the same cDNA source has been used on other primer pairs and they returned gel products that agree with the primers product size. My primers have been designed such that forward and reverse primers are in separate exons, I couldn't get the primers to span exon-intron junctions. I have even had some primer pairs return bad efficiency values yet product sizes are valid; so I am struggling to think if primer design is at fault here as my method of primer design has worked for my other genes.
Does anyone know what potentially could be causing this? Could it be a simple case of primer design? Any answers will be greatly appreciated.
Many thanks in advance.
if you let us know which genes are a problem it might be possible to see if these are members of a similar gene family or have associated pseudogenes or alternatively spliced genes
- I agree with Paul: as your primers are situated internally within exins alternative splice forms or pseudo genes could be the issue
- I would look again at both ref Seq transcript models in GenBank but also Ensemble:
- Then design more primers and situate them in exons (if not spanning exons) common to all transcripts identified in both Ensembl and GenBank models
Paul Rutland, many thanks for your answer. The genes I am looking at are BGLAP and ALOX12 for pigs. The mRNA sequence for ALOX12 for pigs is currently listed as "predicted" on NCBI, so I guess this could be causing difficulties with the product size. BGLAP, however, is stumping me as there is a splice variant of BGLAP (X1) however if the primers have bound this then this should also return a product size similar to the original gene.
Laurence Hall, many thanks for your answer. I will explore Genebank and Ensembl further. It looks like I might have to re-design my primers but I will take what you told me into consideration.
Thanks once more for your answers.
I had a look for similar genes but no luck although all of my experience is in human genomes. Having found nothing the only other thing that I can think of is that one primer has homology with a common repeat and the product is part of a repeat sequence so it might be worth doing a pcr with one primer only to see if this is really an artefact. Naturally this would also need your cDNA to be genomic contaminated so is unlikely, Of course sequencing the long product will tell you a lot about it.
sorry not much help
paul
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