Author Identifier (ORCID)
Elin S. Gray: https://orcid.org/0000-0002-8613-3570
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
Volume
15
Issue
6
Publication Title
Biomolecules
Publisher
MDPI
School
Centre for Precision Health / School of Medical and Health Sciences
RAS ID
82300
Funders
Medical Research Future Fund (2035430) / United States Department of Defense / Melanoma Institute Australia / Tour De Cure / National Health and Medical Research Council / University of Sydney Medical Foundation
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