Before I ask my question, I have to acknowledge that I have very basic knowledge in bioinformatics and I am not a bioinformatician.
I performed a blast search against the sequence of my interest in order to find if there are other variants of the sequence and the results was pretty interesting to me. The blast was showing that about the first 113 bp and last 200 bp of my sequence is occurring in 95 different strains of the same species. Next, I downloaded fasta sequences for all these sequences and ran MSA (WebPRANK and MAFFT). Unlike my initial expectations (which was something like if A=B A=C, then B=C), the pattern of similarity was quite different to blast search, for example the first 82 nucleotides was repeating in all sequences not 113 (this also slightly varied in between different MSA tools).
My question is which method and tool should I use to find out the exact conserved nucleotides?
I think conservation depends on the species (or strains) you are looking for (e.g conserved from yeast to human). For that you have to set sequences from different species/strains that make sense (in the yeast example, for instance, yeast, fly, mouse and human) and construct a MSA with these sequences. In this way you are reconstructing the evolutive history of your sequence of interest and you can assess, in a more robust way, conservation of specific nucleotides.
I don't post this as an answer because I am not an expert on this issue. However, if is helpful I could post it as an answer latter.