Real-time PCR method for detection of short DNA using a deoxyuridine probe and application for detection of fomivirsen

This study sought to develop a short DNA detection method using a deoxyuridine probe and polymerase chain reaction. The probe was hybridized to the target short DNA, which was then extended by DNA polymerase. The extended DNA was used for real-time PCR after the probe was removed by uracil DNA glycosylase. This method measured from 0.01 to 10 nM of a model short DNA sequence of 17 nucleotides. The method was then used to detect the nucleic acid medicine fomivirsen, as well as 21 phosphorothioate nucleotides, and to quantify 0.1–100 nM of fomivirsen. This method may be useful for detecting short DNA fragments, such as functional nucleotides. The PCR method in this study detected short DNA via a simple two-step process. First, the target short DNA was hybridized to a deoxyuridine probe and was extended by DNA polymerase. Second, the extended DNA was mixed with real-time PCR reagent containing uracil DNA glycosylase and then was analyzed by real-time PCR.