Dear researchers,
As you know, it might be some ambiguity nucleotides in primer oligos appeared. For example, in my case, which I took the forward and reverse primers from a paper, there are some of them.
Froward: GGTGGTGTMGGDTTCACMCARTA
Reverse: CGTTCATBGCGTAGTTVGGRTAGT
Now, I would like to know, what a exact nucleotide among the normal nucleotides (A, G, C and T) places inseated of the ambiguity ones (M, R, V, D and B) when a primer making center generates them?
Thanks in advance
Mehrdad Rabiei
When you make oligos with a degeneracy just submit the sequence to the company exactly as you have done here. the company will make oligos with all/both of the degenerate bases at each position so when your sequence says M then you will get a mixture of A and C equally in 2 oligos. If the next degeneracy is D then they will make 3 oligos with AG or T at this position and so on. If you have 4 degeneracies each of 2 possible bases then the product they send you is actually 16 different oligos in the mixture. Oligos are often designed this way when amplifying a mixture of species all with different sequences at the primer position
When you have many degeneracies the amount of any single sequence is very small so pcr conditions may need to be adjusted
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