Ligation-Based qPCR-Amplification Assay for Radiolabel-Free Detection of ATP and NAD⁺ with High Selectivity and Sensitivity

Abstract

We have developed a new sensing system based on quantitative real-time polymerase chain reaction assay (qPCR) to detect adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+) with high sensitivity and selectivity. T4 DNA ligase can catalyze the ligation of two short oligonucleotides (DNA1 and DNA2), which complement a template (cDNA), in the presence of its cofactor, ATP, resulting in increased template concentration and decreased Ct values in qPCR assays. Similarly, the Escherichia coli DNA ligase is also able to catalyze the ligation of DNA1 and DNA2 upon the addition of NAD+. Moreover, this approach has potential for detecting other important cofactors in related systems. Therefore, as a convenient and sensitive strategy, the method may light new beacons and find broad application in biological fields