芯片瞬间等速电泳-筛分电泳偶联分析精确计算DNA长度

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

建立了基于相对迁移时间比例的方法,依据待侧DNA片段相对于上位及下位内标的迁移时间比例进行长度预测。实验结果显示,DNA片段的相对迁移时间比例在不同分析条件下具有良好的重现性。通过建立相对迁移时间比例相对于DNA片段长度的对应关系公式,实现了芯片瞬间等速电泳条件下DNA片段长度的准确预测。实际样品分析证实这种基于相对迁移时间比例的计算方法简单可靠,适合于芯片tITP-CGE分析中DNA长度的精确判定。

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.