I want to design some taq man assays to distinguish between different splice variants of genes. However some of these splice variants have pretty small nucleotide differences such as an extra 47 nucleotides in one of them. When i use the primer3 software to design my primers and probe for the alternative spliced region I get back a result which gives me a probe that is very close to the splice site, so close in fact that only 4 nucleotides of the probe are specific to the one variant i want to detect while the other 16 nucleotides are also present in another splice variant.
My question is - will the 4 nucleotide difference be sufficient to give me specificity to the one splice variant. The four nucleotide specificity is at the 5' end of the probe.
I would calculate the Tm for the 20-mer (4 + 16) versus the 16-mer and see what the difference is. In theory you should be able to do it using the proper annealing temperature. Did you have the software give you all possible primer pairs?
Are you going to use the Roche probes?, since their primer3 software designs primers based on their probes and that limits the primer design.
Having an additional 47 nucleotides should be rather useful, as you can place one of the primers into that region and get a highly specific assay without the need for a probe. Of course it depends on the sequence, but it should be possible to design such an assay. With regards to your probe problem, using an LNA probe should help you - but four nucleotides ought to be sufficient to allow you to discriminate between targets. If you can afford it, then Allele ID (Premier Biosoft) allows you to design specific assays. If you like, you can send me the sequences you want to discriminate and I can see what Allele ID comes up with.
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