{"doi":"10.1093/clinchem/hvae086","title":"Pyrimidine-Dependent UV-Mediated Cross-Linking Magnifies Minor Genetic or Epigenetic Changes in Clinical Samples","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.","journal":"Clinical Chemistry","year":2024,"id":473704,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9583,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1309632,"name":"Farzana Ahmed","orcid":"0000-0003-2019-671X","position":1,"is_corresponding":false},{"id":1309633,"name":"Stavroula Smilkou","orcid":"0000-0002-5338-0276","position":2,"is_corresponding":false},{"id":372743,"name":"Sayeda Yasmin‐Karim","orcid":null,"position":3,"is_corresponding":false},{"id":933981,"name":"Farzaneh Darbeheshti","orcid":"0000-0002-4957-9004","position":4,"is_corresponding":false},{"id":725941,"name":"Athina Markou","orcid":"0000-0001-7760-9701","position":5,"is_corresponding":false},{"id":923992,"name":"Martyn Bullock","orcid":"0000-0002-3740-5509","position":6,"is_corresponding":false},{"id":552333,"name":"Ioannis Boukovinas","orcid":"0000-0003-4475-1802","position":7,"is_corresponding":false},{"id":38080,"name":"Viktor A. Adalsteinsson","orcid":"0000-0003-4555-2485","position":8,"is_corresponding":false},{"id":725942,"name":"Evi Lianidou","orcid":"0000-0002-7796-5914","position":9,"is_corresponding":false},{"id":401885,"name":"G. Mike Makrigiorgos","orcid":"0000-0001-7598-2406","position":10,"is_corresponding":false},{"id":403289,"name":"Fangyan Yu","orcid":null,"position":0,"is_corresponding":true}],"reference_count":43,"raw_metadata":null,"created_at":"2026-07-19T02:06:04.967429Z","pmid":"39039866","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}