Author Identifier
Elin S. Gray: https://orcid.org/0000-0002-8613-3570
Document Type
Journal Article
Publication Title
Biomolecules
Volume
15
Issue
6
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
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.
DOI
10.3390/biom15060883
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