Gene mutations are important biomarkers for the diagnosis, classification, monitoring, and prognosis evaluation of cancers and genetic diseases. Both personalized cancer treatment and noninvasive prenatal testing require methods to accurately determine the abundance of mutation. At present, the widely adopted and convenient methods for measuring mutation abundance are mainly based on relative quantification, which requires negative samples and strict control of the analyte amounts. The development of DNA-probe-based methods that can determine the mutation abundance without negative samples nor control of analyte amount is highly preferred. The key to solving this bottleneck lies in whether the probe’s response to mutation abundance can be completely independent of the number of targeted DNA strands. Herein, we propose the design of a self-internal-reference probe system. We established a theoretical model of this system and used the model to guide the design of probes. In this model, we provided quantitative corrections to the test results from the internal reference, thereby eliminating the influence of substrate amount. Therefore, the purification and quantification processes toward polymerase chain reaction (PCR) amplicons can be omitted. We applied this system to analyze unquantified PCR products aimed at cancer mutation detection and noninvasive prenatal testing.

中文翻译：

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用于突变丰度的无控制定量的自内参探针系统

基因突变是癌症和遗传疾病的诊断、分类、监测和预后评估的重要生物标志物。个性化癌症治疗和无创产前检测都需要方法来准确确定突变的丰度。目前，广泛采用且方便的突变丰度测量方法主要基于相对定量，这需要阴性样本和严格控制分析物的量。开发基于 DNA 探针的方法可以确定突变丰度，无需阴性样本，也无需控制分析物的量。解决这个瓶颈的关键在于探针对突变丰度的反应是否可以完全独立于目标DNA链的数量。在此处，我们建议设计一个自内部参考探针系统。我们建立了该系统的理论模型，并用该模型指导探针的设计。在该模型中，我们对来自内参的测试结果进行了定量修正，从而消除了底物量的影响。因此，可以省略针对聚合酶链反应 (PCR) 扩增子的纯化和量化过程。我们应用该系统分析未量化的 PCR 产物，旨在癌症突变检测和无创产前检测。因此，可以省略针对聚合酶链反应 (PCR) 扩增子的纯化和量化过程。我们应用该系统分析未量化的 PCR 产物，旨在癌症突变检测和无创产前检测。因此，可以省略针对聚合酶链反应 (PCR) 扩增子的纯化和量化过程。我们应用该系统分析未量化的 PCR 产物，旨在癌症突变检测和无创产前检测。