Base modification and cloning efficiency of oligodeoxyribonucleotides synthesized by the phosphoramidite method: methyl versus cyanoethyl phosphorus protection.
We have found large oligodeoxyribonucleotides (50-120 bases) synthesized with N,N-dialkylmethylphosphoramidites to have considerably lower cloning efficiencies than biological DNA. As evidenced by HPLC analysis, these large fragments contain as much as 30% thymidine conversion to 3-methylthymidine. If cyanoethyl- instead of methyl-phosphorous protection is employed, as anticipated, no thymidine methylation is noted. More importantly, large fragments produced by N,N-diisopropylcyanoethyl-phosphoramidite coupling show cloning efficiencies indistinguishable from biological DNA.