Nam Hwanhee, Lee Esder, Yang Hichang, Lee Kyeyoon, Kwak Taeho, Kim Dain, Kim Hyemin, Yang Mihwa, Yang Younjoo, Son Seungwan, Nam Young-Hyean, Minn Il
Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, United States.
NuriBio Co., Ltd, Anyang-si, Gyeonggi-Do, 14058, Republic of Korea.
Biol Methods Protoc. 2024 Jun 4;9(1):bpae041. doi: 10.1093/biomethods/bpae041. eCollection 2024.
Real-time polymerase chain reaction (real-time PCR) is a powerful tool for the precise quantification of nucleic acids in various applications. In cancer management, the monitoring of circulating tumor DNA (ctDNA) from liquid biopsies can provide valuable information for precision care, including treatment selection and monitoring, prognosis, and early detection. However, the rare and heterogeneous nature of ctDNA has made its precise detection and quantification challenging, particularly for ctDNA containing hotspot mutations. We have developed a new real-time PCR tool, PROMER technology, which enables the precise and sensitive detection of ctDNA containing cancer-driven single-point mutations. The PROMER functions as both a PRObe and priMER, providing enhanced detection specificity. We validated PROMER technology using synthetic templates with known KRAS point mutations and demonstrated its sensitivity and linearity of quantification. Using genomic DNA from human cancer cells with mutant and wild-type KRAS, we confirmed that PROMER PCR can detect mutant DNA. Furthermore, we demonstrated the ability of PROMER technology to efficiently detect mutation-carrying ctDNA from the plasma of mice with human cancers. Our results suggest that PROMER technology represents a promising new tool for the precise detection and quantification of DNA containing point mutations in the presence of a large excess of wild-type counterpart.
实时聚合酶链反应（实时PCR）是一种在各种应用中对核酸进行精确定量的强大工具。在癌症管理中，对液体活检中循环肿瘤DNA（ctDNA）的监测可为精准医疗提供有价值的信息，包括治疗选择与监测、预后评估以及早期检测。然而，ctDNA的稀有性和异质性使其精确检测和定量颇具挑战，尤其是对于含有热点突变的ctDNA。我们开发了一种新的实时PCR工具——PROMER技术，它能够精确且灵敏地检测含有癌症驱动单点突变的ctDNA。PROMER兼具探针和引物的功能，可提高检测特异性。我们使用具有已知KRAS单点突变的合成模板对PROMER技术进行了验证，并证明了其定量的灵敏度和线性。利用来自具有突变型和野生型KRAS的人类癌细胞的基因组DNA，我们证实PROMER PCR能够检测突变DNA。此外，我们展示了PROMER技术有效检测患有人类癌症的小鼠血浆中携带突变的ctDNA的能力。我们的结果表明，PROMER技术是一种很有前景的新工具，可在存在大量野生型对应物的情况下精确检测和定量含有单点突变的DNA。
