I am working to amplify a sample with coinfected similar RNA virus to check their competitive fitness. after viral rna extraction, then proceed to cdna synthesis. later i going to amplify both the viruses using a pair of primers that will target the consensus sequence of that two viruses, and later differentiate them using RE cut (the RE cutting site only present in one virus while absent in another).
now, the question is, how am i make sure that the primers will not selectively to amplify certain virus type. and any idea to minimize this if the bias really occur?
As you mentioned that these two viruses has the consensus sequence and you can identify them based up on the amplicon size after PCR amplification.
Hi Antonio,
in your case some bias will inevitably happen (for reasons why see the paper attached below). To minimize the bias try to use greater primer concentrations, longer annealing and elongation times. The concentration of input RNA also makes sense - lower copy number of your targets will lead to greater bias.
But before starting your multitemplate experiment you have to check the influence of PCR protocol variations with different variants of the target separately
Hi
I agree with the above and you can attempt an in vitro PCR analysis by taking the two viral templates in different ratios and see if one is more efficiently amplifying than the other in the PCR reaction. Subsequently, you digest the PCR product and quantify the uncut and digested bands. It may not be perfect experiment but will certainly provide some results if gross differences are there...
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