An Optimization Generator of Synthetic DNA Fragments for the Rational Design of Environmental Tracers.

Chemically synthesized DNA fragments are increasingly recognized as highly valuable tracers for investigating environmental pollution due to their inherent high specificity, sequence diversity, environmental friendliness, stable migration, and high detection sensitivity, outperforming traditional ion and dye tracers. Despite their advantages, a systematic approach for generating suitable DNA sequences, which is a critical requirement for preparing DNA tracers, remains not fully developed. This study introduces an optimization generator of synthetic DNA sequences guided by seven principles, which enables the concurrent generation of multiple sequences with enhanced stability, specificity, and detectability. The DNA sequences produced by our optimization generator display a balanced base distribution, uniform melting temperatures, and reduced formation of hairpin and dimer structures. The necessity of the established principles was further validated through PCR and qPCR detection, showing that noncompliance led to unstable or undetectable DNA amplification. The column and sandbox injection experiments also demonstrated that the generated DNA sequences can be clearly distinguished and effectively used for hydrological multitracing applications. Our research underscores the importance of established principles in creating suitable DNA sequences and offers valuable insights for the efficient preparation of DNA tracers.