次等位基因频率
表观遗传学
DNA
等位基因
遗传学
生物
辅修(学术)
分子生物学
基因
等位基因频率
政治学
法学
作者
Fangyan Yu,Farzana Ahmed,Stavroula Smilkou,Sayeda Yasmin-Karim,Farzaneh Darbeheshti,Athina Markou,Martyn Bullock,Ioannis Boukovinas,Viktor A. Adalsteinsson,Evi Lianidou,G. Mike Makrigiorgos
标识
DOI:10.1093/clinchem/hvae086
摘要
Abstract Background Detection of minor DNA allele alterations is becoming increasingly important for early detection and monitoring of cancer. We describe a new method that uses ultraviolet light to eliminate wild-type DNA alleles and enables improved detection of minor genetic or epigenetic changes. Methods Pyrimidine-dependent UV-based minor-allele enrichment (PD-UVME) employed oligonucleotide probes that incorporated a UVA-sensitive 3-cyanovinylcarbazole (CNVK), placed directly opposite interrogated pyrimidines, such as thymine (T) or cytosine (C) in wild-type (WT) DNA. Upon UVA-illumination, CNVK cross-linked with T/C, preventing subsequent amplification. Mutations that removed the T/C escaped cross-linking and were amplified and detected. Similarly, CNVK discriminated between methylated and unmethylated cytosine in CpG dinucleotides, enabling direct enrichment of unmethylated DNA targets. PD-UVME was combined with digital droplet PCR (ddPCR) to detect serine/threonine-protein kinase B-Raf (BRAF) V600E mutations in model systems, thyroid patient cancer tissue samples, and circulating DNA of tumor origin (ctDNA) from melanoma patients. Results One thyroid cancer sample out of 9, and 6 circulating-DNA samples out of 7 were found to be BRAF V600E-positive via PD-UVME while classified as negative by conventional ddPCR. Positive samples via conventional ddPCR were also found positive via PD-UVME. All 10 circulating cell-free DNA (cfDNA) samples obtained from normal volunteers were negative via both approaches. Furthermore, preferential enrichment of unmethylated alleles in MAGEA1 promoters using PD-UVME was demonstrated. Conclusions PD-UVME mutation/methylation enrichment performed prior to ddPCR magnifies low-level mutations or epigenetic changes and increases sensitivity and confidence in the results. It can assist with clinical decisions that hinge on the presence of trace alterations like BRAF V600E.
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