The Role of Amplicon Length and Total DNA Length in Synthetic DNA Tracer Degradation and Adsorption During Transport Through Sand

Abstract

Synthetic DNA tracers are increasingly used in subsurface hydrological studies because their large sequence diversity enables the design of numerous uniquely identifiable tracers. However, previous studies have not isolated how amplicon length, flanking region length, and total DNA length independently control tracer degradation and adsorption during subsurface transport. In this study, eight double‐stranded DNA (dsDNA) tracers with systematically varied amplicon and total sequence lengths were designed to disentangle the effects of these dsDNA tracer design parameters. Batch degradation experiments were conducted in two types of waters to determine degradation rates, and column transport experiments in two types of sand media were performed to evaluate adsorption behavior. Adsorption and degradation losses during transport were separated using calibrated kinetic sorption models. Results show that degradation rates of dsDNA tracers are primarily controlled by amplicon length rather flanking region length. Tracers with identical amplicon lengths but different flanking region lengths exhibited similar degradation rates, whereas tracers with longer amplicons degraded significantly faster. In contrast, adsorption behavior was governed by the total DNA length, with adsorption increasing linearly with sequence length in both Ottawa sand ( y  = 0.0013 x  + 0.370, R ²  = 0.934) and finer quartz sand ( y  = 0.0006 x  + 0.724, R ²  = 0.999), while detachment rate constants decreased linearly with increasing length. These findings demonstrate that degradation and adsorption are controlled by the dsDNA tracer design parameters and provide a mechanistic basis for designing tracers with predictable transport behavior in subsurface hydrological systems.