Direct Detection of Low Abundance Genes of Single Point Mutation

Area of Science:

• Biotechnology
• Molecular Biology
• Nanotechnology

Background:

• Cell-free DNA (cfDNA) analysis, particularly circulating tumor DNA (ctDNA), offers noninvasive cancer detection opportunities.
• Current PCR-based methods for ctDNA analysis face limitations in sensitivity and specificity.
• There is a need for novel, highly sensitive methods for early cancer assessment.

Purpose of the Study:

• To develop and validate a direct detection method for KRAS G12D mutated genes in clinical ctDNA samples.
• To achieve superior limit of detection (LOD), sensitivity, and specificity compared to existing methods.
• To explore the potential of force-based atomic force microscopy (AFM) for ctDNA analysis.

Main Methods:

• Immobilization of MutS protein onto the tip of an atomic force microscope (AFM).
• Utilizing the MutS protein to sense mismatched sites in DNA duplexes formed between a surface-bound capture probe and target DNA.
• Direct detection of KRAS G12D mutated genes in clinical ctDNA samples.

Main Results:

• Achieved a noteworthy limit of detection (LOD) of 3 copies (0.006% allele frequency).
• Demonstrated superb sensitivity (100%) and specificity (100%).
• Validated the efficacy of force-based AFM with MutS protein and designed probes for ctDNA analysis.

Conclusions:

• Force-based AFM, utilizing MutS protein and optimized probes, represents a novel and highly effective approach for ctDNA analysis.
• This method offers significant improvements in LOD, sensitivity, and specificity for detecting cancer-associated mutations.
• The technique holds promise as an exciting new avenue for noninvasive early cancer assessment.