Synergistic enhanced rolling circle amplification based on mutS and radical polymerization for single-point mutation...

Area of Science:

• Biochemistry
• Molecular Biology
• Nanotechnology

Background:

• Nucleic acid detection in biofluids is crucial for disease diagnosis, but low concentrations necessitate highly sensitive methods.
• Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, common in non-small cell lung cancer, are point mutations requiring precise detection.
• Existing methods like rolling circle amplification (RCA) lack the selectivity and sensitivity for circulating tumor DNA.

Purpose of the Study:

• To develop a highly sensitive and selective method for detecting KRAS mutations.
• To improve the detection limit for circulating tumor DNA.
• To enable early and accurate diagnosis of non-small cell lung cancer.

Main Methods:

• Utilized rolling circle amplification (RCA) for DNA amplification.
• Enhanced RCA selectivity using the mutS enzyme for specific point mutation binding.
• Employed atomic transfer radical polymerization (ATRP) to amplify weak signals from RCA.
• Integrated mutS, RCA, and ATRP with electrochemical sensors for detection.
• Performed computational and experimental analyses to validate the detection steps.

Main Results:

• Achieved high sensitivity and selectivity for KRAS mutation detection.
• Improved RCA selectivity using mutS to specifically target point mutations.
• Reduced detection time by performing RCA and ATRP simultaneously.
• Established a calculated detection limit as low as 3.09 attomolar (aM).
• Validated the method using normal human serum samples.

Conclusions:

• The combined mutS-RCA-ATRP approach with electrochemical sensors offers a sensitive and selective platform for KRAS mutation detection.
• The method demonstrates significant potential for the early diagnosis of cancer patients.
• This technique addresses the limitations of current methods for detecting low-abundance circulating tumor DNA.