Advanced Ligase\nChain Reaction Strategy to Generate\na Circular DNA Walker for Electrochemiluminescent Detection of Single\nNucleotide Polymorphism

Single nucleotide polymorphism (SNP) primarily refers\nto DNA sequence\npolymorphism caused by variations in a single nucleotide, which is\nclosely associated with many diseases such as genetic disorders and\ntumors. However, trace DNA mutants typically exist in a large pool\nof wild-type DNA, making it challenging to establish accurate and\nsensitive approaches for SNP detection. Herein, we developed an advanced\nligase chain reaction (LCR) strategy to output the circular DNA walker\nfor signal amplification, which realized accuracy and sensitive SNP\ndetection based on the electrochemiluminescent (ECL) platform. Unlike\nthe general LCR system that utilizes two sets of short single-stranded\nDNA (ssDNA) primers to generate double-stranded DNA amplification\nproducts, we ingeniously designed a long single-stranded DNA primer\nto replace one set of short ssDNA primers, allowing for the generation\nof circular DNA products upon complementing the target. Noticeably,\nthe circular DNA serves as a DNA walker that can be easily purified\nby nucleases to eliminate unreacted primers and byproducts, significantly\nimproving accuracy and sensitivity. Then, the circular DNA walker\nmoved along a linearly ordered DNA quenching probe track modified\non the ECL sensing interface, restoring the ECL signals by cleaving\nthe quenching probes labeled on the DNA track with the help of apurinic/apyrimidinic\nendonuclease 1. Employing the p53 gene as a model, we realized the\nsensitive detection of mutant p53 in the range from 10 aM to 10 pM,\nwith a detection limit of 6 aM, providing a promising platform for\nSNP detection.