Ribonucleotide-Modified Primer Overlapping Blocker Elongation (PROBE) Assay for Highly Specific Mutation Detection

Single-nucleotide variants (SNVs) are the most common type of genetic variation and are associated with some diseases. Accurate detection of SNVs is of great significance in multiple fields. Despite the rapid development of SNV detection technologies, most methods suffer from limitations in detection specificity, which may lead to false positive results, especially for low-frequency SNVs. Here, we report a novel mutation detection method, the ribonucleotide-modified primer (r-primer) overlapping blocker elongation (PROBE) method. The PROBE method combines RNase H2-mediated r-primer-specific cleavage and blocker-mediated blocking to achieve selective amplification of the mutant (MUT) templates and efficient suppression of wild-type (WT) template amplification. The PROBE method requires allele-specific (AS)- and non-AS-r-primers, along with one peptide nucleic acid (PNA) or locked nucleic acid (LNA) blocker, all of which are blocked by a 3′-C3 spacer. The AS-r-primer and the blocker completely match the MUT and WT, respectively. They overlap and competitively bind to the SNV-containing region. The PROBE method can completely inhibit WT amplification, achieving a sensitivity of 200 copies per reaction and a selectivity of 0.1%. We developed PROBE assays for single-tube discrimination of homozygous (WT and MUT) and heterozygous genotypes of alcohol dehydrogenase 1B (ADH1B) rs1229984 and aldehyde dehydrogenase 2 (ALDH2) rs671 polymorphisms. Clinical evaluation revealed that the PROBE assay achieved accuracies of 100% for rs1229984 and 99.1% for rs671, with the latter slightly exceeding 98.1% accuracy of Sanger sequencing.