Artificial base mismatches-mediated PCR (ABM-PCR) for detecting clinically relevant single-base mutations.

Area of Science:

• Molecular biology
• Genetics
• Biotechnology

Background:

• Accurate detection of single-base mutations is critical for cancer diagnosis and treatment.
• Existing methods face challenges in achieving high sensitivity and specificity.
• Novel approaches are needed for simple, economical, and ultrasensitive mutation detection.

Purpose of the Study:

• To develop and validate an artificial base mismatches-mediated PCR (ABM-PCR) approach.
• To enable ultrasensitive and ultraspecific detection of single-base mutations.
• To provide a simple and economical detection method for clinical applications.

Main Methods:

• ABM-PCR was integrated with quantitative PCR (qPCR) and droplet digital PCR (ddPCR).
• The study examined the impact of primer-template mismatches on PCR amplification.
• Genotyping performance was characterized based on mismatch sequence, position, and number.

Main Results:

• ABM-PCR successfully detected clinically relevant EGFR and BRAF mutations.
• The method achieved 0.1% mutation detection sensitivity with no wild-type amplification.
• Ultrasensitive (≥95%) and ultraspecific (≥95%) diagnosis was demonstrated for lung and thyroid cancers.

Conclusions:

• Primer-template mismatches significantly influence PCR amplification.
• Rational design of ABM-PCR primers enables high specificity and sensitivity for mutation detection.
• ABM-PCR is a valuable tool for clinical diagnosis and prognosis.