Acr-PNAcyp4-mediated method precisely detects KRAS SNPs at 0.66 fM without thermal cycling

Accurate and sensitive detection of single nucleotide polymorphisms (SNPs) is critical for disease diagnosis and personalized medicine. Traditional methods often require sophisticated thermal cycling equipment, limiting their use in clinical and resource-limited settings. This creates a gap for simpler, highly specific, and sensitive techniques. Peptide nucleic acids (PNAs) offer high specificity and stability, making them ideal candidates for developing advanced diagnostic tools that can overcome these limitations.

Researchers developed a fluorescent method for highly specific single nucleotide polymorphism (SNP) detection in mixed-sequence KRAS double-stranded DNA (dsDNA). The core involved selective dsDNA opening using an acridine-linked cyclopentane PNA (Acr-PNAcyp4), followed by padlock probe hybridization and rolling circle amplification (RCA). Resulting RCA products were detected via PNA molecular beacon (PNA MB) probes. The method was validated for detection limits and discrimination capability, then applied to KRAS mutation detection in colorectal cancer cells.

The novel method achieved precise detection of target dsDNA at concentrations as low as 0.66 fM, demonstrating high sensitivity. It enabled effective discrimination among different mutation types in KRAS dsDNA. The high specificity was attributed to PNA-mediated strand invasion (PMSI) and ligation reactions, combined with the robust amplification of RCA technology.

The approach successfully detected KRAS mutations in colorectal cancer cells, confirming its practical utility in complex biological samples. Crucially, this method does not require sophisticated thermal cycling equipment, simplifying the diagnostic process and making it suitable for resource-limited environments. This represents a significant advancement for detecting low-abundance disease-related mutations.