We believe that the expanded genetic alphabet enabled us to eliminate many of the steps that are required in technologies based on just the two base pairs provided by nature. The steps eliminated include solid support washings, transfers, aspirations, and high-temperature hybridizations. Specifically, we believe that there are many reasons that the extra base pair is beneficial. For example, we used short code sequences in the capture step to decode the complex extension mixtures. This may be possible with code sets constructed from only A, G, C, or T, but optimization would surely be more difficult because of cross-hybridization with sequence complements or close complements found in nature. The additional informational content that iC:iG builds into DNA also allows for greater diversity when compared with their all-natural counterparts. Specifically, if the four naturally occurring bases are used to make a library of 10mers, there are 4 10 , or 10 6 possibilities. In contrast, a six-letter system generates 6 10 , or 10 8 unique 10mers. As demon-Clinical Chemistry 50, No. 11, 2004 strated here, specific assembly can occur at conditions such as room temperature, which is in effect more amenable to and simplifies molecular diagnostics.