Precise Prediction of Deoxyribonucleic Acid sizes with Transient Isotachophoreis-Capillary Gel Electrophoresis Analysis on a Microchip / 分析化学

ABSTRACT

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