Wei Yen Chan, Ashleigh Stewart, Russell J Diefenbach, Elin S Gray, Jenny H Lee, Richard A Scolyer, Georgina V Long, Helen Rizos
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引用次数: 0
摘要
循环肿瘤DNA (ctDNA)是一种很有前途的个体化肿瘤生物标志物。然而,其临床应用受到检测灵敏度的限制,特别是在早期疾病中。T-Oligo引物聚合酶链式反应(TOP-PCR)是一种商业扩增方法,利用高效的“半适配器”连接设计和基于单引物的PCR策略。本研究评价了无细胞DNA (cfDNA)预扩增技术的临床应用价值。使用TOP-PCR扩增cfDNA,保留了DNA大小谱,由于半接头连接,cfDNA大小增加了22 bp。基因靶扩增率各不相同,富含gc的TERT启动子扩增子效率较低,而BRAF和TP53扩增子效率较高。优化的预扩增(20 ng cfDNA输入和5-7个PCR循环)提高了ctDNA检测的灵敏度,并扩大了检测多种肿瘤知情突变的样本可用性。重要的是,PCR错误出现在预扩增的cfDNA样本中,强调了阴性对照和建立严格的突变阳性阈值的必要性。
Pre-Amplification of Cell-Free DNA: Balancing Amplification Errors with Enhanced Sensitivity.
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.
BiomoleculesBiochemistry, Genetics and Molecular Biology-Molecular Biology
CiteScore
9.40
自引率
3.60%
发文量
1640
审稿时长
18.28 days
期刊介绍:
Biomolecules (ISSN 2218-273X) is an international, peer-reviewed open access journal focusing on biogenic substances and their biological functions, structures, interactions with other molecules, and their microenvironment as well as biological systems. Biomolecules publishes reviews, regular research papers and short communications. Our aim is to encourage scientists to publish their experimental and theoretical results in as much detail as possible. There is no restriction on the length of the papers. The full experimental details must be provided so that the results can be reproduced.