Abstract
•Novel PEFS-based multi-channel ME for HTS-specific identification of the ACE I/D polymorphism.•About 300 times faster than conventional slab gel electrophoresis (<25s).•PCR products detectable after only 10 PCR cycles.•Fast and reproducible ME method for HTS of DNA with a defined length.
An insertion (I)/deletion (D) polymorphism in angiotensin-converting enzyme (ACE) has been associated with susceptibility to various diseases in numerous studies. Traditionally, slab gel electrophoresis (SGE) after polymerase chain reaction (PCR) has been used to genotype this ACE I/D polymorphism. In this study, single- and multi-channel microchip electrophoresis (ME) methods based on variable programmed electric field strength (PEFS) (i.e., low constant, high constant, (+)/(−) staircase, and random electric field strengths) were developed for fast high-throughput screening of this specific polymorphism. The optimum PEFS conditions were set as 470V/cm for 0–9s, 129V/cm for 9–13s, 470V/cm for 13–13.9s, 294V/cm for 13.9–16s, and 470V/cm for 16–20s for single-channel ME, and 615V/cm for 0–22.5s, 231V/cm for 22.5–28.5s, and 615V/cm for 28.5–40s for multi-channel ME, respectively. In the multi-channel PEFS-ME, target ACE I/D polymorphism DNA fragments (D=190bp and I=490bp) were identified within 25s without loss of resolving power, which was ∼300 times faster than conventional SGE. In addition, PCR products of the ACE gene from human blood samples were detected after only 10 cycles by multi-channel PEFS-ME, but not by SGE. This parallel detection multichannel-based PEFS-ME method offers a powerful tool for fast high-throughput ACE I/D polymorphism screening with high sensitivity.