Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5′ and 3′ ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg–20 ng linear detection range between thermal cycle threshold (C_t value) and target content. The C_t values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology.

中文翻译：

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用于 DNA 计算答案确定的挂锁探针介导的 qRT-PCR。

挂锁探针介导的实时定量 PCR (PLP-qRT-PCR) 适用于量化连续 10mer DNA 序列的丰度，用于 DNA 计算以确定旅行商问题的最佳答案。该方案包括： (i) 线性 PLP 与目标 DNA 序列的杂交；(ii) 通过酶促连接的 PLP 环化；(iii) 去除非环化模板后环化 PLP 的 qRT-PCR 扩增。线性 PLP 设计为由位于 5' 和 3' 端的两个 10 聚体序列检测臂组成，由通用 PCR 引物和 qRT-PCR 报告基因结合位点组成的核心序列隔开。每个 PLP 分子的环化取决于与目标序列的杂交和高保真连接。因此，PLP 环化的数量由溶液中靶标的丰度决定。PLP 的扩增效率在热循环阈值 (C_t值）和目标内容。来自三个目标的多重 qRT-PCR的 C _t值与用单重检测获得的值没有显着差异。该协议为同时量化多个短核酸序列提供了一种高度灵敏和有效的方法，在生物技术中具有广泛的应用。