“ These are not subtle technical disagreements – but fundamental methodological failures – that render the study’s conclusions invalid for regulatory assessment. ”
BUFFALO, NY – August 25, 2026 – A new precision oncology paper was published in Volume 17 of Oncotarget on August 14, 2026, titled “ Systematic methodological flaws in DNA contamination assessment of mRNA vaccines: A critical analysis of Achs et al. (2025) .”
The article was led by first and corresponding author Kevin McKernan from Medicinal Genomics, Beverly, Massachusetts, along with co-authors David J. Speicher from Cyrus Scientific Inc, Hamilton, Ontario, Canada , and Jessica Rose from Brownstone Institute, Austin, Texas .
Rather than presenting a new experimental vaccine analysis, the paper critically examines the methodology used by Achs et al. in a 2025 study that reported no excessive residual DNA impurities in COVID-19 mRNA vaccines. McKernan and colleagues argue that several methodological choices in that study could systematically underestimate residual DNA and therefore limit its suitability for regulatory safety assessment.
One major concern involves how quantitative PCR results were converted from DNA copy numbers into mass. Achs et al. used full-length plasmid molecular weight in their calculations even though their own sequencing data suggested much shorter median DNA fragment sizes. The critique argues that this approach requires fragmentation-correction factors because random DNA breakage can disrupt qPCR target regions and reduce the number of detectable amplicons. Without such correction, the authors contend that residual DNA mass may be underestimated.
The paper also highlights the importance of primer and amplicon design. Achs et al. used qPCR targets with substantially different amplicon lengths, including shorter kanamycin-resistance targets and longer spike-encoding targets. Because the reported median DNA fragment sizes were approximately 130–201 base pairs, longer amplicons would be less likely to remain intact after fragmentation. The authors therefore argue that this design could preferentially reduce detection of spike-associated DNA relative to shorter plasmid regions.
Another concern centers on sample preparation for sequencing. According to the critique, heating samples at 95°C for 10 minutes before Illumina library preparation could further fragment DNA before its native size distribution is measured. The authors contrast this approach with Oxford Nanopore long-read sequencing, which can analyze DNA without PCR amplification and can detect substantially longer fragments. They argue that short-read library preparation, heat treatment, and amplification may collectively bias fragment-size estimates toward shorter DNA molecules.
The analysis also questions whether some analytical platforms used by Achs et al. had sufficient sensitivity to evaluate DNA concentrations near regulatory limits. The paper notes that the Fragment Analyzer configuration cited in the study had a reported lower detection limit of approximately 50 pg/μL for DNA smears, whereas a 10-ng-per-dose threshold distributed across a 300-μL dose corresponds to approximately 33.3 pg/μL. The authors argue that this creates a measurement range in which samples could exceed the stated threshold while remaining below the instrument’s detection limit.
A further issue raised in the paper concerns the vaccine matrix itself. The authors argue that components such as salts, polyethylene glycol, and cationic lipid nanoparticles could interfere with electrokinetic injection during capillary electrophoresis. Because standards were reportedly prepared in clean buffer rather than a matched vaccine matrix, the critique contends that matrix effects could produce lower apparent DNA signals in vaccine samples than in calibration standards.
The paper also focuses on the possibility of RNA:DNA hybrid formation during in vitro transcription. The authors cite previous work indicating that DNase I has substantially lower activity against RNA:DNA hybrids than against double-stranded DNA and argue that such hybrids could protect residual DNA sequences from degradation. They therefore contend that failure to specifically measure RNA:DNA hybrids represents an important gap in residual DNA assessment.
“ Proper assessment of vaccine DNA impurities requires methodologies specifically designed to detect and quantify the biologically relevant forms of residual DNA, including RNA:DNA hybrids and long fragments, using analytical platforms with appropriate sensitivity .”
Beyond these central issues, the authors discuss additional concerns involving fragment-size recovery during DNA extraction, the use of short-read sequencing for long-fragment detection, fluorometric quantification, manufacturing-process differences, and the need for matrix-matched controls. They propose that a more rigorous analytical framework should combine fragment-appropriate qPCR calculations, optimized amplicon design, preparation methods that minimize thermal degradation, long-read sequencing, sensitive quantitative platforms, and direct measurement of RNA:DNA hybrids.
Importantly, this article is a methodological critique, not an independent experimental replication of vaccine DNA measurements. Its conclusions reflect the authors’ interpretation of the methods and data reported by Achs et al. The paper argues that the cumulative effect of the identified biases could systematically underestimate residual DNA, but resolving the disagreement will ultimately require further experimental work using validated methods capable of accurately measuring fragment size, concentration, matrix effects, and RNA:DNA hybrid content.
Overall, the precision oncology paper challenges the analytical foundation of a study that has been cited as evidence that residual DNA levels in mRNA vaccines fall within accepted limits. By examining qPCR mass conversion, fragmentation bias, sample preparation, analytical sensitivity, sequencing-platform limitations, and RNA:DNA hybrids, the authors call for more rigorous and transparent methods before firm regulatory conclusions are drawn about residual DNA contamination.
DOI: https://doi.org/10.18632/oncotarget.28913
Correspondence to: Kevin McKernan – Kevin.McKernan@medicinalgenomics.com
Keywords : mRNA vaccines, DNA contamination, qPCR, plasmid DNA, RNA:DNA hybrids
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Systematic methodological flaws in DNA contamination assessment of mRNA vaccines: A critical analysis of Achs et al. (2025)
14-Aug-2026
DS and JR declare no conflicts of interest. KM is employed at Medicinal Genomics, which manufactures qPCR reagents for agricultural applications.