Novel Primer Design for Significantly Reducing Fluorescent Interferences in the Synthesis of DNA-Templated Copper Nanoclusters for the Detection of the HLA-B*5801 Gene

The optimal sequence for synthesizing copper nanoclusters is a promising research area. Initially, random dsDNA sequences yielded low fluorescence intensity, which constrained visual detection under UV light. Poly-AT dsDNA sequences later produced visible fluorescence, but it caused significant interference in negative samples when combined with gene amplification techniques. This interference occurs because the single-stranded poly-AT primer can self-anneal into a double-stranded AT sequence, efficiently synthesizing copper nanoclusters. To mitigate this, we designed a poly-AAT sequence at the primer’s 5′ end, creating a single base pair mismatch every three nucleotides during self-annealing. This adjustment reduced synthesis efficiency of copper nanoclusters in negative samples, improving the visual distinction between negative and positive results. We applied this method to identify the HLA-B*5801 gene, thereby demonstrating its efficacy even within a GC-rich region of human genomic DNA. Our method showed 100% agreement with a commercial qPCR kit, with results distinguishable under UV light. We conclude that the poly-AAT sequence is more suitable for integrating copper nanoclusters synthesis with nucleic acid amplification detection techniques, with potential applications in microelectronics, biosensing, and catalysis.