I've identified a novel splice variant, and want to develop an assay to distinguish it from the other known variant. The only region I can target is small (the variant has a different splice site that excludes 23 bp from following exon of the known variant). Given that it is not just the only differing exon boundary, but the only region I can prime against to distinguish the two variants, does anyone have any tips for actually running the protocol? BLAST suggests only one primer in that region, and it has a low Tm (57.76 degrees) and low GC (36%). It's not a great place to start a qPCR experiment, but I'd like to know if it is still feasible. Before I move forward, can people suggest how to improve efficiency for such a primer? Or what the low end of acceptability is for primers in qPCR?
The calculations of Tm are still performed by algorithms that are not perfect - so, you have every confidence going forward at this point knowing that you have a possibility in the first place. Some primers that shouldn't work very well at all work like a charm. Algorithms are not all-knowing yet at this point regarding Tm values... they are getting better - but it depends on which algorithms you ultimately use, and what ionic strength you are working at. Also, primer concentration itself directly affects their effective Tm as well...
Hi Nicholas,
This paper might be helpful - it describes how you can design hairpin primers that have very exquisite discrimination between SNPs. It may be helpful for discriminating between your two splice variants.
Good luck
Mark
Article Exquisite allele discrimination by toehold hairpin primers
Article A reliable method for quantification of splice variants using RT-qPCR
This might be helpful !
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