Throughout the history of oligonucleotide synthesis, there are reports of modification of guanine at the 6-position. This modification has been apparent in both solution- and solid-phase phosphotriester chemistry as well as in solid-phase phosphoramidite chemistry. The most effective way to eliminate this modification may be to protect the 6-position of·guanine. However, the additional cost of fully-protected 2'-deoxyGuanosine monomers may limit their acceptance for routine DNA synthesis.
Despite much speculation on the cause and nature of the modification at the 6-position of guanine, no formal study had been carried out. However, recently two researchers at Applied Biosystems, Inc. reported1 that the initial event in the process of modification of guanine is the phosphitylation of the oxygen at the 6-position. This adduct is partially cleaved by acetate ion in the capping mix but, after iodine oxidation, a more stable phosphotriester species is formed. In the capping step of a subsequent cycle, the phosphotriester adduct is displaced by 4,4-dimethylaminopyridine (DMAP) (present in the capping mix) to form a fluorescent species. Ammonia in the deprotection step leads to the substitution of a portion of the DMAP containing adducts to form 2,6- diaminopurine, albeit in relatively low levels.
Following an exhaustive series of experiments, the authors are able to offer a solution to the problem of modification of the 6-position of guanine. By substituting N-methylimidazole (Melm) for DMAP as the capping catalyst, the presence of fluorescent impurities and 2,6- diaminopurine in the product sequences is virtually eliminated.
A recent report2 describes an oxidant with a significantly improved shelf life for automated oligonucleotide synthesis. The most popular oxidant used in phosphoramidite chemistry is 0.1 M 12 in THF/2,6-lutidine/H2O (40:10:1 ). For the most part this solution has proved to be effective and stable. However, solutions which are exposed to the atmosphere may develop a black oily residue. This problem may be alleviated by the use of 0.05M 12 in THF/Water/Pyridine (7:2:1 ). This solution has proved to be effective in DNA synthesis.
1. J.S. Eadie and D.S. Davidson, Nucleic Acids Res., 1987, 15, 8333.
2. R.T. Pon, Nucleic Acids Res., 1987, 15, 7203.
10% 1-Methylimidazole in Tetrahydrofuran (40-4120), Also, please see our complete list of Capping Reagents.
0.05M Iodine in Tetrahydrofuran/ Water/ Pyridine (40-4130) has been discontinued. Please see our complete list of Oxidation Reagents.