Reactions of buffers in cyanogen bromide-induced ligations.

Heike Vogel, Claudia Gerlach, Clemens Richert

Index: Nucleosides Nucleotides Nucleic Acids 32(1) , 17-27, (2013)

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Abstract

Rapid, template-directed ligation reactions between a phosphate-terminated oligonucleotide and an unphosphorylated reaction partner may be induced by cyanogen bromide (BrCN). Frequently, however, the reaction is low yielding, and even a large excess of the condensing agent can fail to induce quantitative conversions. In this study, we used BrCN to induce chemical primer extension reactions. Here, we report that buffers containing hydroxyl groups react with short oligodeoxynucleotides in the presence of BrCN. One stable adduct between HEPBS buffer and cytosine was characterized by mass spectrometry and NMR after HPLC purification, indicating that a side reaction occurred at this nucleobase. Further, a first example of a primer extension reaction between an unmodified oligodeoxynucleotide as primer and dGMP is reported. Together, our results shed light on the potency, as well as the drawbacks of BrCN as a highly reactive condensing reagent for the ligation of unmodified nucleic acids.