Single Nucleotide Discrimination by alpha-Hemolysin Nanopore

摘要

We in this paper presented a single-base resolution method for discrimination of single base pair differences within DNA duplexes containing A-T matched base pairs and A-C mismatched base pairs using alpha-hemolysin (alpha-HL) protein nanopore embedded in a lipid bilayer. A 17-mer probe respectively annealed to two 57-mer target ssDNA which had poly(dT)20 tails on both 5' and 3' ends, forming single base-pair differentiating DNA duplexes containing the matched A-T base pairs and A-C mismatched base pairs which were then voltage-driven captured and stacked into the alpha-HL constriction zone under a biased potential. After a long unzipping process, the longer stranded and probe DNA was successively separated and translocated through the alpha-HL channel, then generating a characteristic signal. Ion channel recordings were used to detect and compare the unzipping process. The duplex structures exhibited different unzipping time duration under applied voltage by firstly threading either the 3' or 5' overhangs into the alpha-hemolysin channel that permitted easy discrimination between the completely matched and single-mismatched base pairs. The simple biosensing strategy will open up a variety of applications on SNP sites detection, diseases classification, medical screening and diagnosis.