Optimized Multi-Phosphorothioate Linkers Enable Efficient Iodine-Induced Cleavage of Surface-Immobilized DNA for Genomic Applications.

Efficient solid-phase DNA cleavage is essential for advanced biotechnology. In this study, we employed multiple phosphorothioate (PT) modifications within the DNA backbone to achieve a cleavage efficiency of >93% under mild conditions on a biochip surface. This method was successfully implemented in a next-generation sequencing (NGS) workflow as a promising alternative to the conventional 8-oxoG/formamidopyrimidine DNA glycosylase (FPG) enzymatic system. By optimizing the PT modification sites and reaction conditions, our platform delivered sequencing data quality, including Q30 scores, single-nucleotide polymorphism (SNP), and insertions and deletions (INDEL) accuracy, equivalent to established standards. Additionally, the mild reaction conditions are compatible with downstream polymerase chain reaction (PCR) amplification, enabling the process to bypass the traditional purification steps. This highly efficient, cost-effective chemical cleavage approach facilitates seamless integration into automated genomic analysis platforms and streamlined molecular diagnostics.