Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the α-Hemolysin Nanopore.

ABSTRACT: The latch region of the wild-type α-hemolysin (α-HL) protein channel can be used to distinguish single base modifications in double-stranded DNA (dsDNA) via ion channel measurements upon electrophoretic capture of dsDNA in the vestibule of α-HL. Herein, we investigated the use of the latch region to detect a nick in the phosphodiester DNA backbone. The presence of a nick in the phosphodiester backbone of one strand of the duplex results in a significant increase in both the blockade current and noise level relative to the intact duplex. Differentiation between the nicked and intact duplexes based on blockade current or noise, with near baseline resolution, allows real-time monitoring of the rate of T3-DNA ligase-catalyzed phosphodiester bond formation. Under low ionic strength conditions c

SUBMITTER: Tan CS 

PROVIDER: S-EPMC5302010 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature