• Park, Kyong Hwa
• Park, Junsoo
• Kim, Yeul Hong
• Abbas, Naseem
• Shin, Sehyun
• Park, Youngsoo

Abstract

Single nucleotide polymorphism (SNP) are frequently observed during anti-cancer therapy and their detection is critical for the prognosis and therapeutic monitoring of cancers. However, the detection of SNP is challenging due to the poor selectivity and limit of detection of the existing clinical evaluation methods because of the extreme similarities between the wild-type oligonucleotides and those with SNP. Therefore, in this study, we developed an innovative method to detect SNP with high sensitivity and selectivity. In the proposed method, LNA-modified primers and hairpin-shaped primers interact synergistically to promote target selectivity and sensitive amplification in real-time polymerase chain reaction (PCR). This method was able to detect the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) point mutation E545K from cell-free DNA with 0.1% selectivity and 10 aM (0.12 fg) limit of detection. The present method was further successfully applied to the clinical samples of a colorectal cancer patient and the results were validated using digital droplet PCR. The present method of utilizing LNA-modified hairpin primers was found to be a highly sensitive, simple, and robust diagnostic tool to detect the PIK3CA E545K point mutation, which can also be used to detect various single nucleotide polymorphism in the patients with cancer.

Topics

• impedance spectroscopy
• additive manufacturing