One-Pot Identification of BCR/ABL^p210 Transcript Isoforms Based on Nanocluster Beacon

The BCR/ABL^p210 fusion gene is a classic biomarker of chronic myeloid leukemia, which can be divided into e13a2 and e14a2 isoforms according to different breakpoints. These two isoforms showed distinct differences in clinical manifestation, treatment effect, and prognosis risk. Herein, a strategy based on nanocluster beacon (NCB) fluorescence was developed to identify the e13a2 and e14a2 isoforms in one-pot. Because the fluorescence of AgNCs can be activated when they are placed in proximity to the corresponding enhancer sequences, thymine-rich (T-rich) or guanine-rich (G-rich). In this work, we explored an ideal DNA-AgNCs template as an excellent molecular reporter with a high signal-to-noise ratio. After recognition with the corresponding isoforms, the AgNCs can be pulled closer to the T-rich or G-rich sequences to form a three-way junction structure and generate fluorescence with corresponding wavelengths. Therefore, by distinguishing the corresponding wavelengths of AgNCs, we successfully identified two isoforms in one tube with the limitation of 16 pM for e13a2 and 9 pM for e14a2. Moreover, this strategy also realized isoform identification in leukemia cells and newly diagnosed CML patients within 40 min, which provides a powerful tool to distinguish fusion gene subtypes at the same time.