The design of primers is frequently complicated by the degeneracy of the genetic code. Three strategies are now available to confront this problem. In the first, a mixed base addition (N) is used to form the degenerate site. This approach is best if the number of degenerate sites is small. A second option is the use of 2'-deoxyInosine or 2'-deoxyNebularine which exhibit low, but unequal, hydrogen bonding to the other four bases. The third option is the use of a universal nucleoside. In this strategy, the base analog does not hybridize significantly to the other four bases and makes up some of the duplex destabilization by acting as an intercalating agent. 3-Nitropyrrole 2'-deoxynucleoside (M) is the first example of a set of universal bases. Subsequently, 5-nitroindole was determined to be an effective universal base and to be superior to 3-nitropyrrole, based on duplex melting experiments.
The modified bases designated P and K show considerable promise as degenerate bases. The pyrimidine derivative P, when introduced into oligonucleotides, base pairs with either A or G, while the purine derivative K base pairs with either C or T. A dP+dK mix also can serve as a mixed base with much less degeneracy than dA+dC+dG+dT (N).
Coupling: No changes needed from standard method recommended by synthesizer manufacturer. Note inosine is somewhat susceptible to damage by iodine during oxidation. If there are >6 incorporations of inosine within the sequence, use 0.5M CSO in anhydrous acetonitrile (40-4632-xx) and a 3 minute oxidation time for best results.
Deprotection: No changes needed from standard method recommended by synthesizer manufacturer.
Deoxy-Inosine is often used as a degenerate base in an oligonucleotide probe or primer. This is possible since the structure allows it to base pair with all four bases in various wobble structures. However the base-pairing is not equivalent with each of the 4 naturally occuring bases. The overall preferential order of base-pairing is. dI-dC > dI-dA > dI-dG = dI-dT||REFERENCE(S):Martin,F.H. et.al., (1985), Nucleic Acids Res., 13, 8927-8938., Case-Green,S. C., Southern, E.M., (1994), Nucleic Acids Res., 22, 131-136.