AMPLON: Amplifying DNA with Multi-Arm Priming and Looping Optimization of Nucleic Acid.

Nucleic acid amplification, the bedrock of biotechnology and molecular diagnostics, surges in applications-especially isothermal approaches-heightening the demand for advanced and precisely engineered methods. Here, we present a novel approach for Amplifying DNA with Multi-arm Priming and Looping Optimization of Nucleic Acid (AMPLON). AMPLON relied on a novel polymeric material that utilizes a unique set of multi-arm polyethylene glycol-DNA primers for efficient DNA amplification under isothermal conditions. Each arm carries ssDNA complementing the sense or antisense sequence of the target DNA (n = 6; 50% sense to 50% antisense sequences). The amplification reaction begins with antisense arms binding to the target DNA, forming a template for sense-carrying arms to direct multi-arm large DNA amplicon synthesis through successive DNA looping and unlooping steps. The developed AMPLON enables highly specific and sensitive detection of the targeted nucleic acid sequence. Using HIV-1 as a model clinical target, AMPLON exhibited high sensitivity, detecting target concentrations as low as 100 copies/mL, and selectively amplifying HIV-1 in the presence of other DNA and RNA viruses, such as HBV and HCV. Compared to a quantitative real-time PCR (qRT-PCR) assay using sensitive primers, AMPLON reliably identifies HIV-1 RNA in plasma samples (n = 20) with a significant agreement rate of 95%. With its ability to achieve highly specific and sensitive target amplification within 30 minutes, AMPLON holds immense potential to transform the field of nucleic acid research and unleashing new possibilities in medicine and biotechnology. This article is protected by copyright. All rights reserved.