Eliminating the secondary structure of targeting strands for enhancement of DNA probe based low-abundance point mutation detection

Abstract

Nucleic acid probes are very useful tools in biological and medical science. However, the essential sensing mechanism of nucleic acid probes was prone to the interference of surrounding sequences. Especially when the target sequences formed secondary structures such as hairpin or quadruplex, the nucleic acid probes were hindered from hybridizing with target strands, greatly disabled the function of probes. Herein, we have established an Open strand based strategy for eliminating the influence of secondary structures on the performance of nucleic acid probes. The strategy was general toward different lengths, secondary structures and sequences of the targeting strand, and we found that the improvement was higher when the secondary structure of the targeting strand was more complicated. Experiments on synthetic single stranded DNA and real clinical genomic DNA samples were conducted for low abundance mutation detection, and the limit of detection for TERT-C228T and BRCA2 rs80359065 mutations could be 0.02% and 0.05% respectively, demonstrating the clinical practicability of our proposed strategy in low abundance mutation detection.

Keywords: Fluorescent detection; Low-abundance detection; Nucleic acid probes; Point mutations; Secondary structure.