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.

中文翻译：

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嘧啶依赖性 UV 介导的交联放大了临床样本中微小的遗传或表观遗传变化


背景 检测微小的 DNA 等位基因改变对于癌症的早期检测和监测变得越来越重要。我们描述了一种使用紫外线消除野生型 DNA 等位基因并能够改进对微小遗传或表观遗传变化的检测的新方法。方法 嘧啶依赖性基于 UV 的次要等位基因富集 （PD-UVME） 采用寡核苷酸探针，该探针掺入了 UVA 敏感的 3-氰基咔唑 （CNVK），直接位于野生型 （WT） DNA 中询问的嘧啶（如胸腺嘧啶 （T） 或胞嘧啶 （C）的对面。在 UVA 照射下，CNVK 与 T/C 交联，阻止了后续扩增。去除 T/C 的突变逃逸交联并被扩增和检测。同样，CNVK 区分 CpG 二核苷酸中的甲基化和未甲基化胞嘧啶，从而能够直接富集未甲基化的 DNA 靶标。PD-UVME 与数字液滴 PCR （ddPCR） 相结合，检测模型系统、甲状腺患者癌症组织样本和黑色素瘤患者肿瘤来源循环 DNA （ctDNA） 中的丝氨酸/苏氨酸蛋白激酶 B-Raf （BRAF） V600E 突变。结果 通过 PD-UVME 发现 9 个甲状腺癌样本中有 1 个，7 个样本中有 6 个循环 DNA 样本为 BRAF V600E 阳性，而通过常规 ddPCR 分类为阴性。通过常规 ddPCR 的阳性样本也通过 PD-UVME 发现阳性。通过两种方法从正常志愿者获得的所有 10 个循环游离 DNA （cfDNA） 样本均呈阴性。此外，使用 PD-UVME 证明了 MAGEA1 启动子中未甲基化等位基因的优先富集。 结论 ddPCR 前进行的 PD-UVME 突变/甲基化富集放大了低水平突变或表观遗传变化，提高了结果的敏感性和可信度。它可以协助临床决策，这些决策取决于是否存在 BRAF V600E 等痕量改变。