A FEN 1-driven DNA walker-like reaction coupling with magnetic bead-based separation for specific SNP detection

Abstract

Single-nucleotide polymorphism (SNP) plays a key role in the carcinogenesis of the human genome, and understanding the intrinsic relationship between individual genetic variations and carcinogenesis lies heavily in the establishment of a precise and sensitive SNP detection platform. Given this, a powerful and reliable SNP detection platform is proposed by a flap endonuclease 1 (FEN 1)-driven DNA walker-like reaction coupling with a magnetic bead (MB)-based separation. A carboxyfluorescein (FAM)-labeled downstream probe (DP) was decorated on a streptavidin magnetic bead (SMB). The target DNA, as a walker strand, was captured by hybridization with DP and an upstream probe (UP) to form a three-base overlapping structure and execute the walking function on the surface of SMB. FEN 1 was employed to specifically recognize the three-base overlapping structure and cut the 5′flap at the SNP site to report the walking event and signal amplification. Considering the fact that the fluorescence was labeled on the cleavage and uncleavage sequences of DP and the target DNA-triggered walking event was undistinguishable from the mixtures, magnetic separation came in handy for cleavage probe (CP) isolation and discrimination of the amplified signal from the background signal. In comparison with the conventional DNA walker reaction, this strategy was coupling with SMB-based separation, thus promising a powerful and reliable method for SNP detection and signal amplification.