Why is it that when a sequence contains a ‘wobble’ it has variable functionality?
Incorporation of 'wobbles' into a sequence decreases the effective concentration of each species. With increased numbers of 'wobbles' the number of distinct species increases exponentially thereby decreasing the likelihood that any individual sequence has the desired specificity. Only one species is usually present in a biological sample. As some portion of the oligo species are not completely complementary to the target, some variability in function is to be expected. To further complicate matters, the individual nucleotide amidites can have different coupling rates. Each time the same 'wobble' sequence is synthesized, there is the potential that one species will be produced in preference over another.
1) Be conservative. Introduce as few 'wobbles' as possible; one trinucleotide 'wobble' and one dinucleotide 'wobble' or two dinucleotide 'wobbles' in two different locations such that there is a maximum of 6 variants in a single oligonucleotide and;
2) Increase the concentration of your 'wobble' sequence by as much as two fold to compensate for the presence of multiple unique sequences.
If you have specific questions regarding minimum yields for a particular probe, please contact our Technical Support