Abstract

Gene point mutations play a significant role in the development of cancer. Therefore, developing a sensitive, specific, and universally applicable method for detecting gene point mutation is crucial for clinical diagnosis, prognosis, and cancer treatment. Recently, gene point mutation detection methods based on CRISPR/Cas12a detection have emerged. However, existing methods generally lack universality and specificity. In this study, we have developed a CRISPR/Cas12a-based method that combines improved allele-specific polymerase chain reaction and single base extension to translate the point mutation information in the target dsDNA into length information in ssDNA activators to overcome the limitations associated with PAM sequences in the CRISPR/Cas12a system. Our method achieved a detection limit of 0.002% for clinically significant EGFR T790M mutation. The CRISPR/Cas12a system we constructed demonstrates high sensitivity, specificity, and universality in detecting gene point mutations, making it a promising tool for clinical cancer screening.

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