Uracil is not always a mistakenly occurring base in DNA. Uracils in DNA genomes are known to be important in the life cycles of Bacillus subtilis phages (PBS1/2) and the malarial parasite, Plasmodium falciparum ; and have been implicated in the development of fruit fly and antibody maturation in B-lymphocytes. Availability of a sensitive, specific and robust technique for the detection uracils in genes/genomes is essential to understand its varied biological roles. Mycobacterium smegmatis UdgX (Msm UdgX), identified and characterised in our laboratory, forms covalent complexes with the uracil sites in DNA in a specific manner. Msm UdgX cleaves the glycosidic bond between uracil and the deoxyribose sugar in DNA to produce uracilate and oxocarbenium ions. The oxocarbenium ion is then captured into a covalent complex by the nucleophilic attack of a histidine side chain of Msm UdgX. Here, we describe the use of a fusion protein, mCherry tagged Msm UdgX (mChUdgX), which combines the property of Msm UdgX to covalently and specifically bind the uracil sites in the genome, with the sensitivity of fluorescent detection of mCherry as a reporter. We show that both the purified mChUdgX and the Escherichia coli cell-extracts overexpressing mChUdgX provide high sensitivity and specificity of detecting uracils in DNA. Image 1 • mCherry tagged Msm UdgX (mChUdgX) has been developed as a specific probe to detect uracil sites in DNA. • mChUdgX overexpressing E. coli cell-lysate is as good in uracil detection as the pure protein. • Gel retardation assays, confocal microscopy and other assays can be performed using mChUdgX to detect uracil sites in DNA. • The techniques are robust, highly specific and reliable for uracil detection in DNA. [ABSTRACT FROM AUTHOR]