Abstract
Natural chemical modifications of nucleic acids play a vital role in life processes. Compared to other epigenetic modifications, there are multiple ways to quantify the methylated derivatives of cytosine. However, simple and convenient methods for detecting and quantifying thymine derivatives are scarce because they are found in tiny quantities in biological systems. Additionally, exploring easy ways to detect these derivatives can also throw light on their biological significance. This manuscript reports a novel strategy to quantify 5-formyluracil (5fU) and 5-hydroxymethyluracil (5hmU). Differences between modified and unmodified bases are accumulated and amplified by arranging phi29 DNA polymerase to repeat through a circular template labeled thymidine. In combination with real-time quantitative rolling circle amplification (RCA), low-abundance thymine oxides can be quantified precisely. The global levels of 5fU and 5hmU were analyzed in different biological samples, using only 40 ng of sample input on a laboratory real-time PCR instrument. The reported strategy was executed hassle-free and, in principle, can be extended to design methods for detecting other epigenetic modifications in nucleotides that are rare in biological systems.