Duplex stability and, therefore, melting temperature can be raised using the modified bases. For replacement of dC with 5-Me-dC, duplex melting temperature is increased by 1.3 º. Improved stacking in this case is believed to be brought about by elimination of water molecules from the duplex. The additional hydrogen bond formed by substituting dA with 2-amino-dA leads to a melting temperature increase of 3º.
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).
A standard Watson and Crick base pair is formed between iso-C and iso-G, but the hydrogen bonding pattern is quite different from the natural base pairs A-T and C-G (The 5-methyl analogue was chosen as the synthetic target due to the reported instability of 2'-deoxyisocytidine caused by deamination during oligonucleotide synthesis or deprotection).
Oligo Purification