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Pre-Amplification of Cell-Free DNA: Balancing Amplification Errors with Enhanced Sensitivity
Wei Yen Chan1,2, Ashleigh Stewart1,2, Russell J Diefenbach1,2
1Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW 2109, Australia.
Biomolecules
|June 26, 2025
Summary
Pre-amplifying cell-free DNA (cfDNA) using TOP-PCR enhances circulating tumour DNA (ctDNA) detection sensitivity. However, careful controls are crucial due to potential PCR errors in amplified cfDNA samples.
Area of Science:
- Molecular Biology
- Oncology
- Biomarker Discovery
Background:
- Circulating tumour DNA (ctDNA) shows promise as a biomarker in personalised oncology.
- Clinical utility of ctDNA is often limited by low detection sensitivity, especially in early-stage cancers.
- Cell-free DNA (cfDNA) pre-amplification is explored to improve ctDNA detection.
Purpose of the Study:
- To evaluate the clinical value and application of cfDNA pre-amplification using T-Oligo Primed Polymerase Chain Reaction (TOP-PCR).
- To assess the impact of TOP-PCR on cfDNA integrity and amplification efficiency for key oncogenes.
- To determine optimal pre-amplification conditions for enhancing ctDNA detection sensitivity.
Main Methods:
- Utilized TOP-PCR, a commercial amplification method with a half-adapter ligation and single-primer strategy, for cfDNA pre-amplification.
- Analyzed cfDNA size profiles post-amplification.
- Assessed amplification efficiency across different gene targets (TERT promoter, BRAF, TP53).
- Optimized pre-amplification parameters including cfDNA input (20 ng) and PCR cycles (5-7).
Main Results:
- TOP-PCR pre-amplification preserved cfDNA size profiles, with a 22 bp increase due to half-adapter ligation.
- Amplification efficiency varied by gene target, with lower efficiency for GC-rich TERT promoter and higher for BRAF and TP53.
- Optimized pre-amplification significantly enhanced ctDNA detection sensitivity and expanded sample utility for multiple mutation detection.
- PCR errors were observed in pre-amplified cfDNA, necessitating rigorous controls.
Conclusions:
- Optimized TOP-PCR pre-amplification improves sensitivity for ctDNA detection, broadening its clinical application.
- The method expands the availability of cfDNA samples for detecting multiple tumour-informed mutations.
- Stringent mutation positivity thresholds and negative controls are essential to mitigate PCR errors in pre-amplified cfDNA.
