Abstrato

Method enabling detection of single-stranded DNA ligation activity.

Tommy Nordstrom, Pal Nyren

The enzyme DNA ligase is an essential tool in many different applications in the field of biotechnology. For these applications the DNA quality can be crucial for the final result. In this paper we present a new method that can be used for specific studies of DNA ligases and for simultaneous analyzes of DNA quality. More specifically, a simple, fast and sensitive method for detection of single-stranded DNA (ssDNA) ligase activity is presented. The new method is based on real-time detection of nucleotide incorporation over the ligation site catalyzed by a DNA polymerase. The ratio between the incorporation signal after and before the ligation site gives an estimate of the ligation efficiency. To further increase the robustness of the assay the exact amount of unligated product can be easily estimated in a separate assay using a 3’-end blocked oligonucleotide functioning as template. Produced circular DNA can be cleaned from unligated DNA by exonuclease I treatment and the quality of the circle can be easily analyzed by a simple sequencing procedure. If the ssDNA used for the ligation reaction contains truncated fragments this will be observed by uneven signals during the analysis. The new method can be a useful tool for researchers working with both basic and applied projects in the field of biotechnology.