# 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

## TL;DR

A new DNA primer design reduces false signals in copper nanocluster synthesis, improving detection of the HLA-B*5801 gene.

## Contribution

A novel poly-AAT primer design minimizes self-annealing interference in DNA-templated copper nanocluster synthesis.

## Key findings

- The poly-AAT primer reduces copper nanocluster synthesis in negative samples, improving visual distinction.
- The method achieved 100% agreement with a commercial qPCR kit for HLA-B*5801 detection.
- The approach works effectively in GC-rich regions of human genomic DNA.

## Abstract

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.