Reduced stutter artifact formation with an engineered thermostable DNA polymerase

Short tandem repeat (STR) profiling is foundational to forensic human identification, but stutter artifacts remain a persistent challenge, particularly for mixture interpretation where elevated stutter peaks can obscure minor contributor alleles or be misinterpreted as genuine contributor alleles. Here we describe the development of Reduced Stutter Polymerase (RSP), an engineered novel thermostable DNA polymerase that substantially reduces stutter artifact formation across forensic STR loci. RSP was engineered by inserting the thioredoxin-binding domain (TBD) from T3 DNA polymerase into Taq DNA polymerase and genetically fusing E. coli thioredoxin to the N-terminus via a flexible linker that enables intramolecular interaction between thioredoxin and TBD. Scanning mutagenesis across the entire TBD and thioredoxin domains identified beneficial mutations that were systematically combined with linker length refinement to yield RSP. When tested using a duplex STR assay targeting trinucleotide repeats, RSP reduced back stutter 5- to 6-fold and forward stutter ~2-fold compared to Taq DNA polymerase. Characterization across mono-, di-, tri-, tetra-, and pentanucleotide repeat structures revealed that back stutter reduction was most pronounced for tri- and tetranucleotide repeats (5- to 8-fold reduction), with dinucleotide repeats showing ~4-fold reduction, pentanucleotide repeats 3- to 4-fold reduction, and more modest improvements for mononucleotide repeats (1.4-fold). Using the Promega PowerPlex® 35GY primer set as a representative multiplex platform, RSP reduced back stutter an average of 8.1-fold across 31 loci in 56 individuals (all loci p < 0.05). RSP also substantially reduced the dependence of stutter on allele length and decreased the variability of stutter values observed at each locus. In an analysis of 22 two-person mixture samples (10:1 ratio) designed to challenge stutter-based filtering, RSP-amplified profiles showed significantly fewer stutter assignment errors (0.3 ± 0.6 per profile) compared to control amplifications (5.6 ± 3.3 per profile, p < 0.001). These results suggest that a polymerase engineered for reduced stutter can improve the discrimination between stutter artifacts and genuine contributor alleles. These findings establish the scientific foundation for future commercial forensic RSP-based STR systems.