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