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 (PfAgo) 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 PfAgo 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.

/pdf/argonaute-integrated-single-tube-pcr-system-enables-e0xjh2mbat.pdf

Argonaute integrated single-tube PCR system enables supersensitive detection of rare mutations

The ability to detect rare mutations has revolutionized diagnosis and monitoring of tumors, but is limited by the shortage of sensitive, cost-effective and high coverage methods for identification of extremely low abundant mutations. Here, we establish a single-tube multiplex PCR system by employing thermophilic Argonaute-derived DNA-guided nuclease for highly efficient rare mutation detection, referred to as A-Star (Argonaute-directed specific target enrichment and detection), that combines the selective cleavage of the wild type DNA in the DNA denaturation step and the followed amplification of mutant DNA during PCR. A-Star enables easy detection and quantitation of rare mutations originally as low as 0.01% in allele frequency with a ⩾ 5500-fold efficiency. We also demonstrate the feasibility of A-Star for detecting oncogenic mutations in complex biological systems such as solid tumors tissues and blood samples. Remarkably, A-Star could achieve the detection of multiple oncogenic genes simultaneously through a simple single-tube reaction. Taken together, our work illustrates a supersensitive and rapid nucleic acid detection system, thereby extending the utility for both research and therapeutic applications.

/pdf/argonaute-mediated-system-for-supersensitive-and-multiplexed-38ocfem4bj.pdf

Argonaute-mediated system for supersensitive and multiplexed detection of rare mutations

The detection of rare polymorphic alleles, especially single nucleotide variations (SNVs), is becoming increasingly relevant for the early diagnosis and monitoring of a variety of tumors. However, the broad clinical applicability of SNV detection has been limited by the low sensitivity, flexible target selection and multiplexing offered by existing SNV detection methods. Here, we developed a simple but efficient single-tube PCR system, referred to as A-Star (Ago-directed specific target enrichment), that takes advantage of the thermophilic nature of Pyrococcus furiosus Argonaute (PfAgo) to specifically cleave wild-type sequences during the DNA denaturation step, leading to progressive and rapid (~ 3 h) enrichment of scarce SNV-containing alleles. This approach enables the detection of rare SNVs at an attomolar sensitivity and can amplify allele fractions as low as 0.01% with an over 6000-fold efficiency. We further validated the A-Star system by multiplex detection of three rare oncogenic genes in complex genetic backgrounds. Finally, we successfully applied A-Star to detect oncogenic mutations in cancer patient tissue and blood samples, indicating that our method holds great promise for the diagnosis of various types of cancer as well as the genotyping of rare alleles for basic research.

/pdf/a-star-an-argonaute-directed-system-for-rare-snv-enrichment-d20t3olbrg.pdf

A-Star, an Argonaute-directed System for Rare SNV Enrichment and Detection

A primerless amplification suitable for enrichment of particular genotype cfDNA which is a one-dimensional material has been developed. This primerless amplification coordinated by two thermostable enzymes of endonuclease and proofreading polymerase, functions as a genotype switch in analyzing cfDNA. The endonuclease digests the wild-typed fragments into mega-primer and discriminately destroys the wild-type DNA alleles. The DNA polymerase proofreads the megaprimer and then extends the mega-primer using the mutant DNA as the template. The prototypes of this technology were applied to two hotspot mutations of APC and EGFR with confirmed by DNA sequencing analysis. Genotype switch was then employed to clinical cfDNA assay targeting PIK3CA. Data from the clinical application suggest its potential in early cancer diagnosis.

Primerless Amplification for Circulating Tumor DNA Assays.

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Primerless Amplification for Circulating Tumor DNA Assays.