PDF(Pubmed)
Abstract：
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, a novel approach for amplifying DNA with multiarm priming and looping optimization of nucleic acid (AMPLON) is presented. AMPLON relies on a novel polymeric material with unique set of multiarm polyethylene glycol-DNA primers for efficient DNA amplification under isothermal conditions. Each arm carries single-stranded DNA complementing the sense or antisense sequence of the target DNA. The amplification reaction begins with antisense arms binding to the target DNA, forming a template for sense-carrying arms to direct multiarm large DNA amplicon synthesis through successive DNA looping and unlooping steps. Using human immunodeficiency virus type 1 (HIV-1) as a model clinical target, AMPLON exhibits high sensitivity, detecting target concentrations as low as 100 copies mL-1. Compared to a quantitative real-time polymerase chain reaction 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 min, AMPLON holds immense potential to transform the field of nucleic acid research and unleashing new possibilities in medicine and biotechnology.
摘要：
核酸扩增,生物技术和分子诊断的基石，应用中的激增-特别是等温方法-提高了对先进和精确工程方法的需求。这里,我们提出了一种通过多臂启动和核酸循环优化（AMPLON）扩增DNA的新方法。AMPLON依赖于一种新型聚合物材料，该材料利用一组独特的多臂聚乙二醇-DNA引物在等温条件下进行有效的DNA扩增。每个臂携带与靶DNA的有义或反义序列互补的ssDNA(n=6;50%有义至50%反义序列)。扩增反应从反义臂与靶DNA结合开始，形成有义携带臂的模板，以通过连续的DNA循环和解环步骤指导多臂大DNA扩增子合成。开发的AMPLON能够对靶向核酸序列进行高度特异性和灵敏的检测。使用HIV-1作为模型临床目标，AMPLON表现出高灵敏度，检测目标浓度低至100拷贝/mL，并在其他DNA和RNA病毒存在下选择性扩增HIV-1，如HBV和HCV。与使用敏感引物的定量实时PCR（qRT-PCR）分析相比，AMPLON以95%的显著一致率可靠地鉴定血浆样品(n=20)中的HIV-1RNA。凭借其在30分钟内实现高度特异性和灵敏的靶标扩增的能力，AMPLON拥有巨大的潜力，可以改变核酸研究领域，并在医学和生物技术领域释放新的可能性。本文受版权保护。保留所有权利。
