homologous amplicon for real time pcr - (Nov/03/2010 )
I am very new to the field of molecular biology and am facing some problems related to Taqman qPCR, it would be great if i cud get sum help.
I have received Taqman assays from the company for two genes that are 80% homologous to each other. I only know the amplicon sequences (sequences that the primers and probes amplify). Of the two one amplicon sequence is 90% similar to the other gene sequence. Is there some way i could find out if the taqman assays are specific.
Am i right that it doesnt matter if the amplicon sequences give a number of blast hits, what is important is that your primers are specific?
Some help would be highly appreciated.
To confirm crossreactivity of the assays I would generate in-vitro RNA transcript and test it with both assays. For example assay targeting gene A should be tested on RNA of gene B, and the assay targeting gene B should be tested on RNA of gene A.
MGB or LNA probes could help specificity of the detection when it is hard to achieve using primers.
PCR bias could be tested by crosstitration of RNA A and B and testing both of the assays.