Ultrahigh sensitive and selective detection of single nucleotide polymorphism using peptide nucleic acid and ribonuclease H assembled DNA amplification (PRADA)

Early diagnosis and monitoring of cancer is the best way to increase the survival rate among patients with cancer. However, the current cancer screening technology is expensive and time-consuming; hence, cancer screening remains challenging. Therefore, developing a relatively inexpensive and high-performance analytical method is necessary. Especially, mutations in KRAS can be characterized as single nucleotide polymorphism mutations. Therefore, discrimination of single nucleotide polymorphism is essential to detect cancer mutations. This study introduces a method with high sensitivity and selectivity of real-time PCR using peptide nucleic acid (PNA) and RNase H II to detect KRAS single nucleotide polymorphism. This method was developed via the fusion of the existing PNA clamping PCR technique and the RNase H-dependent PCR technique. A synergistic effect was realized by mitigating the shortcomings of each method. Our method had a detection limit of 1 aM and selectivity of 0.01%. This study demonstrated completed validation tests with DNA-spiked plasma sample analysis, cell culture, and analysis of blood samples collected from patients with cancer. Furthermore, we demonstrated the applicability of this method for breath biopsy.

Automated summary

This study developed a method using PNA and RNase H II for real-time PCR to detect KRAS single nucleotide polymorphism. The method exhibited high sensitivity and selectivity, and achieved a synergistic effect by combining the advantages of both techniques. Validation tests with different samples showed the potential of this method in detecting cancer mutations and its applicability in breath biopsy.