Shaheer H. Khan

Dye terminator chemistry : Effects of substrate structure on sequencing analysis

Dye-labeled dideoxy terminators are the preferred reporter groups for DNA sequencing. This labeling technique can result in uneven peak heights in the electropherograms due to sequence context effects on the biochemical distribution of extension vs termination events. Large variations in peak heights can result in difficulty in identifying very small peaks adjacent to big peaks. Using substrate structure to minimize the sequence context effects on dye-terminator incorporation, we have studied the effects of combinations of base, dye, and dye linkage on terminator incorporation during sequencing. We have identified a propargyl ethoxyamino linker that proved useful in improving peak height evenness and enhancing the terminator activity. New dye-linker-base combinations were optimized for overall peak evenness and relative electrophoretic mobility shifts. The more even peak patterns improve base calling and heterozygote analysis using dye terminator chemistry.