Hi there,
I have design a 20bp forward primer which located at 2 nucleotides before TaqMan probe. The question is that, is spacing of 2 nucleotides enough for polymerase to sit on 3' end of forward primer? Will it affect the amplification of assay?
Hi Lee,
You will often see people saying that there is a minimum gap of 5 bases required but there does not appear to be any evidence for this. In fact, technologies such as MGB probe assays recommend short gaps (1-2 bases) to decrease amplicon size which has many beneficial effects
I have tested dozens of assays with just a single base gap between primer and probe and have not seen an issue. This is similar with many of the 'rules of primer/probe design' such as ideal GC content, avoiding runs of bases, not starting/ending on specific bases etc - they are fine as a guide but don't let them stop you for ordering the optimal assay as they rarely have the effect people believe. Always go with the best option and let the validation show you if it works
I don't think it is recommended to have fewer than 5nt between them.
http://www.sciencedirect.com/science/article/pii/S0167701202001136
https://www.researchgate.net/post/Is_it_necessary_to_have_a_minimum_distance_from_the_primer_in_Taqman
Hi Lee,
You will often see people saying that there is a minimum gap of 5 bases required but there does not appear to be any evidence for this. In fact, technologies such as MGB probe assays recommend short gaps (1-2 bases) to decrease amplicon size which has many beneficial effects
I have tested dozens of assays with just a single base gap between primer and probe and have not seen an issue. This is similar with many of the 'rules of primer/probe design' such as ideal GC content, avoiding runs of bases, not starting/ending on specific bases etc - they are fine as a guide but don't let them stop you for ordering the optimal assay as they rarely have the effect people believe. Always go with the best option and let the validation show you if it works
- Badges
- Science method