Detection of Single Nucleotide Polymorphisms of Circulating Tumor DNA by Strand Displacement Amplification Coupled with Liquid Chromatography

The detection of multiple single nucleotide polymorphisms (SNPs) of circulating tumor DNA (ctDNA) is still a great challenge. In this study, we designed enzyme-assisted nucleic acid strand displacement amplification combined with high-performance liquid chromatography (HPLC) for the simultaneous detection of three ctDNA SNPs. First, the trace ctDNA could be hybridized to the specially designed template strand, which initiated the strand displacement nucleic acid amplification process under the synergistic action of DNA polymerase and restriction endonuclease. Then, the targets would be replaced with G-quadruplex fluorescent probes with different tail lengths. Finally, the HPLC-fluorescence assay enabled the separation and quantification of multiple signals. Notably, this method can simultaneously detect both the wild type (WT) and mutant type (MT) of multiple ctDNA SNPs. Within a linear range of 0.1 fM-0.1 nM, the detection limits of BRAF V600E-WT, EGFR T790M-WT, and KRAS 134A-WT and BRAF V600E-MT, EGFR T790M-MT, and KRAS 134A-MT were 29, 31, and 11 aM and 22, 29, and 33 aM, respectively. By using this method, the mutation rates of multiple ctDNA SNPs in blood samples from patients with lung or breast cancer can be obtained in a simple way, providing a convenient and highly sensitive analytical assay for the early screening and monitoring of lung cancer.