Fast screening of rice knockout mutants by multi-channel microchip electrophoresis

Abstract

A multi-channel microchip electrophoresis (MC-ME) system with a laser-induced fluorescence detector was developed for the fast simultaneous detection of rice knockout mutants in genetically modified (GM) rice. In addition, three parallel separation channels were fabricated on a glass microchip to investigate the possibility of high-throughput screening of amplified-polymerase chain reaction products representing wild-type rice and mutants. The MC-ME system was developed to simultaneously record data on all channels using specifically designed electrodes for an even distribution of electric fields, an expanded laser beam for excitation, a 10× objective lens to capture emissions, and a charge coupled device camera for detection. Under a programmed electric field strength and a sieving gel matrix of 0.7% poly(ethylene oxide) (Mr=8,000,000), T-DNA-inserted rice mutants, two standard wild-type rice lines, and six rice knockout mutants were analyzed within 4min using three parallel channels on the microchip. Compared to conventional microchip electrophoresis, the MC-ME method is a valid and practical way to effectively analyze multiple samples in parallel for the identification of GM rice without any loss of resolving power or reproducibility. The MC-ME method was more than 15 times faster than traditional slab gel electrophoresis and proved to be a powerful tool for high-throughput screening of GM rice with high sensitivity, efficiency, and reproducibility.