SIMPLE, EFFICIENT PROTOCOL FOR ENZYMATIC-SYNTHESIS OF UNIFORMLY C-13,N-15-LABELED DNA FOR HETERONUCLEAR NMR-STUDIES

The use of uniformly C-13,N-15-labeled RNA has greatly facilitated str uctural studies of RNA oligonucleotides by NMR, Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly la beled with C-13 and/or N-15 have been published, but have not yet been widely used. We have developed a modified procedure for preparing uni formly C-13,N-15-labeled DNA based on enzymatic synthesis using Taq DN A polymerase. The highly efficient protocol results In quantitative po lymerization of the template and similar to 80% incorporation of the l abeled dNTPs, Procedures for avoiding non-templated addition of nucleo tides or for their removal are given. The method has been used to synt hesize several DNA oligonucleotides, including two complementary 15 ba se strands, a 32 base DNA oligonucleotide that folds to form an intram olecular tripler and a 12 base oligonucleotide that dimerizes and fold s to form a quadruplex, Heteronuclear NMR spectra of the samples illus trate the quality of the labeled DNA obtained by these procedures.