Pre-amplification of cell-free DNA: Balancing amplification errors with enhanced sensitivity

Authors/Creators

• Wei Yen Chan
• Ashleigh Stewart
• Russell J. Diefenbach
• Elin S. Gray, Edith Cowan UniversityFollow
• Jenny H. Lee
• Richard A. Scolyer
• Georgina V. Long
• Helen Rizos

Author Identifier (ORCID)

Elin S. Gray's ORCID record

Abstract

Circulating tumour DNA (ctDNA) is a promising biomarker for personalised oncology. However, its clinical utility is limited by detection sensitivity, particularly in early-stage disease. T-Oligo Primed Polymerase Chain Reaction (TOP-PCR) is a commercial amplification approach utilising an efficient “half-adapter” ligation design and a single-primer-based PCR strategy. This study evaluated the clinical value and application of cell-free DNA (cfDNA) pre-amplification. cfDNA amplification with TOP-PCR preserved DNA size profiles and resulted in a 22 bp size increase due to the half-adaptor ligation. Gene target amplification rates varied, showing lower efficiency for the GC-rich TERT promoter amplicon and higher efficiency for the BRAF and TP53 amplicons. Optimised pre-amplification (20 ng cfDNA input and 5–7 cycles of PCR) enhanced ctDNA detection sensitivity and expanded sample availability for the detection of multiple tumour-informed mutations. Importantly, PCR errors emerged in pre-amplified cfDNA samples, underscoring the necessity for negative controls and the establishment of stringent mutation positivity thresholds.

Keywords

circulating tumour DNA, detection sensitivity, melanoma

Document Type

Journal Article

Date of Publication

6-1-2025

Article Number

883

Volume

15

Issue

6

Publication Title

Biomolecules

Publisher

MDPI

School

Centre for Precision Health / School of Medical and Health Sciences

RAS ID

82300

Funding Information

W.Y.C. is supported by the CLEARbridge Foundation, Melanoma Institute Australia PhD scholarship, and Tour De Cure Postgraduate Research Grant. R.A.S. and J.H.L. are supported by a National Health and Medical Research Council of Australia (NHMRC) Investigator Grant. G.V.L. is supported by an NHMRC investigators Grant and the University of Sydney Medical Foundation. R.J.D. is supported by the CLEARbridge Foundation. This work was funded by the CLEARbridge Foundation, Medical Research Future Fund (2035430), and United States Department of Defense.

Funding received from the Medical Research Future Fund (MRFF)

2035430

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 License.

Comments

Chan, W. Y., Stewart, A., Diefenbach, R. J., Gray, E. S., Lee, J. H., Scolyer, R. A., Long, G. V., & Rizos, H. (2025). Pre-amplification of cell-free DNA: Balancing amplification errors with enhanced sensitivity. Biomolecules, 15(6), 883. https://doi.org/10.3390/biom15060883

Recommended Citation

Chan, W. Y., Stewart, A., Diefenbach, R. J., Gray, E. S., Lee, J. H., Scolyer, R. A., Long, G. V., & Rizos, H. (2025). Pre-amplification of cell-free DNA: Balancing amplification errors with enhanced sensitivity. Biomolecules, 15(6), Article 883. https://doi.org/10.3390/biom15060883