Direct Quantification of Trace Amounts of a Chronic Myeloid Leukemia Biomarker Using Locked Nucleic Acid Capture Probes

Molecular monitoring is indispensable for the clinical management of chronic myeloid leukemia (CML) patients. Real-time quantitative polymerase chain reaction (RTqPCR) is the gold standard for the quantitative assessment of BCR-ABL transcript levels, which are critical in clinical decision-making. However, the frequent recurrence of the disease after drug discontinuation for 60% of patients has necessitated more sensitive and specific techniques to detect residual BCR-ABL transcripts. Here, we describe a quantification method for the detection of BCR-ABL targets at very low concentrations (<10 copies/sample) in the presence of a million copies of normal BCR and ABL genes. In this method, a fully modified locked nucleic acid (LNA) and a LNA/DNA chimera were used as capture probes, and the quantitative imaging mode of atomic force microscopy (AFM) was employed. Targets with one of the major breakpoints (found in more than 95% of CML patients), b3a2 and b2a2, were quantified. The BCR-ABL target captured on a miniaturized LNA-probe spot was scanned at nanometric resolution, and the samples containing one to ten copies of the BCR-ABL genes were examined. It was observed that the highest sensitivity, i.e., the detection of a single copy of the target gene, could be achieved through multiple runs, and the observed cluster number was well correlative (adjusted R-2 = 0.999) to the target copy number in the sample solution. This observation clearly demonstrates that the LNA-based platform is effective in quantifying BCR-ABL targets with extremely low copy numbers, highlighting the potential applicability of AFM for use in the direct quantification of such targets without amplification or labeling.