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。我们描述了一种新方法，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。依赖于嘧啶的紫外线微小等位基因富集 (PD-UVME) 采用寡核苷酸探针，其中包含 UVA-敏感的 3-氰基乙烯基咔唑 (CNVK)，直接放置在被询问的嘧啶对面，例如野生型 (WT) DNA 中的胸腺嘧啶 (T) 或胞嘧啶 (C)。在 UVA 照射下，CNVK 与 T/C 交联，防止随后的扩增。去除 T/C 的突变逃脱了交联，并被放大和检测到。同样，CNVK 可以区分 CpG 二核苷酸中的甲基化和非甲基化胞嘧啶，从而能够直接富集非甲基化 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 突变通过 PD-UVME 发现 9 份甲状腺癌样本中的 1 份和 7 份循环 DNA 样本中的 6 份为 BRAF V600E 阳性，而通过传统 ddPCR 分类为阴性。通过常规 ddPCR 获得的阳性样本通过 PD-UVME 也发现呈阳性。通过这两种方法从正常志愿者获得的所有 10 个循环游离 DNA (cfDNA) 样本均为阴性。此外，还证明了使用 PD-UVME 优先富集 MAGEA1 启动子中的非甲基化等位基因。在 ddPCR 之前进行的 PD-UVME 突变/甲基化富集可放大低水平突变或表观遗传变化，并提高结果的灵敏度和可信度。它可以协助根据 BRAF V600E 等微量改变的存在做出临床决策。© 诊断协会

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.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.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.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.© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.