Precise Prediction of DNA Sizes with Transient Isotachophoresis-Capillary Gel Electrophoresis Analysis on a Microchip

The migration time in transient isotachophoresis (tITP) separation is affected by sample salinity due to the dependence of ITP time on sample-zone conductivity. The sample-to-sample variation of migration time in microchip tITP-capillary gel electrophoresis (CGE) analysis is an undesired factor for precise DNA sizing. In this study, a DNA-sizing method based on relative migration-time proportion (RMP) was developed to eliminate the effect of sample salinity on sizing precision. RMP is defined as the ratio of the migration-time difference between the target fragment and the lower marker to that of the migration-time difference between the upper marker and the lower marker. The RMP values were tested to be reproducible in microchip tITP-CGE separations irrespective of sample salinity. Size of a target DNA was predicted by placing its RMP value in the equation derived from RMPs of standard DNA ladder vs. DNA sizes. The precision and reproducibility of the sizing method were validated by testing multiple standard PCR amplicons. Experimental results showed that the RMP method was simple and reliable, thus well suited to precise DNA sizing with microchip tITP-CGE technique.