Fluorometric detection of single-nucleotide mutations using tandem gene amplification

Abstract

Sensitive and accurate identification of single-nucleotide mutations (SNMs) has become crucial in the field of molecular diagnosis and personalized medicine. Here, we developed a highly sensitive fluorometric assay for detecting multiple SNMs by tandem gene amplification; reverse transcription PCR (RT-PCR) coupled with rolling circle amplification generating G-quadruplex (GQ-RCA). In tandem gene amplification, RT-PCR-amplified DNA from sample RNA was digested with lambda exonuclease, which degraded the strand harboring the phosphorylated 5′-end of amplicon DNA to generate single-stranded DNA (ssDNA). The resulting ssDNA was hybridized with padlock probe DNA, which can discriminate a single base mismatch. Depending on the presence of mismatched bases between the amplicon ssDNA and padlock probe DNA, ligation of both ends of the padlock DNA was evaluated. If ligation occurred, a circular form of padlock DNA was generated, which was eligible for RCA. The RCA process generates long stretches of ssDNA containing tandem repeats of G-quadruplex structures. The amplified ssDNA harboring G-quadruplex was fluorescently visualized and quantified using Thioflavin T fluorophore. Our assay detected mutant RNA containing an SNM as low as 8.3 fg and detected mutant RNA with a concentration as low as 0.53 % in the mixture with wild-type RNA. The fluorometric SNM detection method with tandem gene amplification was successfully applied for multiplex detection of SNMs in clinical samples derived from patients with chronic myeloid leukemia. Our method would be useful in clinical diagnosis for early detection and monitoring of multiple SNMs in cancer patients with proper treatment.