PROMER 技术:一种新的实时 PCR 工具,能够以高特异性和高灵敏度对点突变进行多重检测。

IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Biology Methods and Protocols Pub Date : 2024-06-04 eCollection Date: 2024-01-01 DOI:10.1093/biomethods/bpae041
Hwanhee Nam, Esder Lee, Hichang Yang, Kyeyoon Lee, Taeho Kwak, Dain Kim, Hyemin Kim, Mihwa Yang, Younjoo Yang, Seungwan Son, Young-Hyean Nam, Il Minn
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引用次数: 0

摘要

实时聚合酶链反应(real-time PCR)是在各种应用中精确定量核酸的有力工具。在癌症治疗中,监测液体活检中的循环肿瘤 DNA(ctDNA)可为精准治疗提供有价值的信息,包括治疗选择和监测、预后和早期检测。然而,ctDNA 的稀有性和异质性使其精确检测和定量具有挑战性,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,它能精确灵敏地检测含有癌症单点突变的 ctDNA。PROMER 同时具有 PRObe 和 priMER 的功能,从而提高了检测的特异性。我们使用已知 KRAS 点突变的合成模板验证了 PROMER 技术,并证明了它的灵敏度和线性定量性。我们使用来自具有突变型和野生型 KRAS 的人类癌细胞的基因组 DNA,证实了 PROMER PCR 可以检测突变 DNA。此外,我们还证明了 PROMER 技术能从人类癌症小鼠的血浆中有效地检测出携带突变的 ctDNA。我们的研究结果表明,PROMER 技术是一种很有前途的新工具,它能在野生型DNA大量存在的情况下精确检测和量化含有点突变的 DNA。
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PROMER technology: A new real-time PCR tool enabling multiplex detection of point mutation with high specificity and sensitivity.

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.

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来源期刊
Biology Methods and Protocols
Biology Methods and Protocols Agricultural and Biological Sciences-Agricultural and Biological Sciences (all)
CiteScore
3.80
自引率
2.80%
发文量
28
审稿时长
19 weeks
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