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This paper presents an experimental and numerical investigation into the use of high-resolution injection techniques to separate DNA fragments within electrophoresis microchips. The study addresses the principal material transport mechanisms such as electrokinetic migration, fluid flow, diffusion, variable-volume injection methods, and gives detail analyses to the double-L injection technique, which employs electrokinetic manipulations to avoid sample leakage within the microchip. We also study the sample leakage effect during sample injection and separation. The standard DNA ladder and the φ-174 DNA fragments are used to test the performance for this proposed method. Results show that this unique injection system in the current microfluidic chip presented within this paper is capable of simulating the functions of the cross, double-T form through appropriate manipulations of the electric field within its various channels. The proposed double-L injection method confirms its ability to reduce sample leakage effect during operation. The integrated microfluidic chip and double-L injection technique developed in this study has an exciting potential for use in high-quality, high-throughput chemical analysis applications and in many other applications throughout the field of micro-total-analysis systems.