I have designed a TaqMan probe with 30 nt in size targeted a segment in mitochondria gene, which is conserved in most vertebrate. Multiple sequence alignment was done. There are still few nucleotides polymorphism (approx 5 single separated bases) in the middle of target sequence.
So I am wondering the whether:
1. Polymorphism will affect the annealing efficiency of PCR?
2. Affect the amplification?
3. Is LNA technology can be utilized to substitute the polymorphic bases?
Share with me any of your thought and it would be greatly appreciated.
Thank you.
first of all Probes r small as 30 bases, secondly try another region of amplicon and design probe where this exactly complementing as taqmans specificity is in its probe
This paper gives some idea of the potential penalties associated with polymorphism away from the specific target
http://www.ncbi.nlm.nih.gov/pubmed/16051376
Hi Sandeep,
In my case, the fully conserved regions has a lower Tm value compare to primers. So I am afraid they probe will not anneal to template before the primer started to amplify. And this definitely make the quantification reaction more risky and less efficient.
I have tried designing in other region but afterall it is difficult for me to come to a compromise between fully conserved region and high Tm value. Although the sequence identity of my current targeted regionis not 100% in all vertebrate, it has a higher Tm value, which is a more promising candidate as a probe. Yet I am just trying to design a primer set that has a broader specificity.
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