The simplest approach to the design of high affinity primers and probes is to substitute A sites with 2-amino-A, since the 2-amino-A-T base pair is equivalent in strength to the G-T base pair. 2-Amino-A also destabilizes A-G wobble mismatches, thus increasing specificity. In 1998, we introduced a 2-amino-dA monomer which exhibits fast and effective deprotection in ammonium hydroxide and it is stabilized to depurination during synthesis. We now recommend the use of 0.5 M CSO in anhydrous acetonitrile (40-4632-xx) for best results with multiple additions of 2-amino-dA. This is because the bis formamidine protected 2-amino-dA leads to significant strand scission when standard iodine oxidation is used during synthesis. For this reason, we have also added Pac-2-Amino-dA, a monomer with optimized protection to meet the following criteria: stable during oligonucleotide synthesis, oxidation, and detritylation; labile towards common deprotection conditions (NH3, AMA, MeNH2); and the nucleobase protecting groups are cleaved under fairly mild conditions.
Coupling: Standard coupling time. Add a maximum of 3 times when using 0.02 M I2 oxidation or use 0.5 M CSO in anhydrous ACN (40-4632-xx) and a 3 minute oxidation time for best results with multiple additions.
Deprotection: Ammonium hydroxide 24h/55°C; AMA 1.5h/65°C or 17h/room temperature.