High dynamic range capillary electrophoresis method for sensitive detection of low-frequency driver mutations.

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Cancer genomics seeks to personalize treatment by identifying genetic alterations.
• Current analytical methods for cancer genomics often lack simplicity and cost-effectiveness.
• Sensitive detection of low-frequency mutations is crucial for early cancer diagnosis and treatment selection.

Purpose of the Study:

• To develop an enhanced capillary electrophoresis (CE) sequencer with improved dynamic range for sensitive cancer mutation detection.
• To evaluate the performance of the HiDy-CE system in detecting low-frequency KRAS mutations in clinical samples.

Main Methods:

• Development of an enhanced capillary gel electrophoresis (CE) sequencer utilizing a High Dynamic range (HiDy) fluorescence-acquisition technique (HiDy-CE).
• Application of the multi-base primer extension method combined with HiDy-CE for mutation detection.
• Validation using control DNA with known mutations and clinical samples from pancreaticoduodenal tumors.

Main Results:

• The HiDy-CE system demonstrated a significantly expanded dynamic range, reducing saturation risk.
• Variant allele frequencies (VAFs) as low as 0.5% for KRAS hotspot mutations were detected.
• HiDy-CE produced VAFs equivalent to targeted amplicon sequencing using only 10 ng of DNA from fine-needle biopsy specimens.
• High concordance with digital PCR was achieved with as little as 2 ng of input DNA, showing minimal non-specific noise.

Conclusions:

• The HiDy-CE system represents a breakthrough in sensitive mutation detection using capillary electrophoresis.
• This technology enables the detection of clinically relevant mutations below 1% in pathological specimens.
• HiDy-CE holds significant potential for sensitive detection of oncogenes like KRAS, facilitating pre-testing for comprehensive genome profiling.