Mutant DNA quantification by digital PCR can be confounded by heating during DNA fragmentation

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Digital PCR (dPCR) is increasingly used for precise DNA mutation quantification in clinical samples.
• Genomic DNA often requires fragmentation before dPCR analysis, but the impact of fragmentation methods is not fully understood.

Purpose of the Study:

• To evaluate the effects of three DNA fragmentation methods (heating, restriction digestion, acoustic shearing) on digital PCR point mutation assay performance.
• To assess the limit of blank (LoB) and gene copy number estimation for each fragmentation technique.

Main Methods:

• Wild-type human genomic DNA was fragmented using heat (95°C), restriction digestion, or Covaris acoustic shearing.
• Digital PCR was employed to quantify wild-type and mutant allele counts for KRAS and BRAF proto-oncogenes.
• Limit of blank (LoB) and gene copy number were determined for each fragmentation method.

Main Results:

• Heat fragmentation at 95°C resulted in a high background mutation frequency for specific KRAS mutations (G>A), likely due to heat-induced DNA damage.
• Heat fragmentation also led to an overestimation of gene copy number, possibly from single-stranded DNA partitioning.
• Covaris acoustic shearing and restriction enzyme digestion demonstrated comparable and reliable LoBs and gene copy number estimates.

Conclusions:

• Heat-induced DNA fragmentation can introduce significant artifacts in digital PCR mutation assays, particularly for G>A mutations.
• Acoustic shearing and restriction digestion are suitable methods for DNA fragmentation prior to dPCR, yielding accurate results.
• Moderate heating during DNA extraction does not appear to significantly affect observed KRAS mutation counts in dPCR.