Argonaute integrated single-tube PCR system enables supersensitive detection of rare mutations. Issue 13 (27th April 2021)

• Record Type: Journal Article
• Authors: Liu, Qian; Guo, Xiang; Xun, Guanhua; Li, Zhonglei; Chong, Yuesheng; Yang, Litao; Wang, Hongxia; Zhang, Fengchun; Luo, Shukun; Cui, Li; Zhao, Pengshu; Ye, Xingyu; Xu, Heshan; Lu, Hui; Li, Xiao; Deng, Zixin; Li, Kai; Feng, Yan
• Is Part Of: Nucleic acids research. Volume 49:Issue 13(2021)
• Page Start: e75
• Page End: e75
• Publication Date: 2021-04-27
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkab274
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: Technological advances in rare DNA mutations detection have revolutionized the diagnosis and monitoring of tumors, but they are still limited by the lack of supersensitive and high-coverage procedures for identifying low-abundance mutations. Here, we describe a single-tube, multiplex PCR-based system, A-Star, that involves a hyperthermophilic Argonaute from Pyrococcus furiosus ( Pf Ago) for highly efficient detection of rare mutations beneficial from its compatibility with DNA polymerase. This novel technique uses a specific guide design strategy to allow Pf Ago selective cleavage with single-nucleotide resolution at 94°C, thus mostly eliminating wild-type DNA in the denaturation step and efficiently amplifying rare mutant DNA during the PCR process. The integrated single-tube system achieved great efficiency for enriching rare mutations compared with a divided system separating the cleavage and amplification. Thus, A-Star enables easy detection and quantification of 0.01% rare mutations with ≥5500-fold increase in efficiency. The feasibility of A-Star was also demonstrated for detecting oncogenic mutations in solid tumor tissues and blood samples. Remarkably, A-Star achieved simultaneous detection of multiple oncogenes through a simple single-tube reaction by orthogonal guide-directed specific cleavage. This study demonstrates a supersensitive and rapid nucleic acid detection system with promising potential for both research and therapeutic applications.