{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T12:28:18Z","timestamp":1786969698628,"version":"build-2736575974"},"reference-count":42,"publisher":"Wiley","issue":"17","license":[{"start":{"date-parts":[[2023,8,21]],"date-time":"2023-08-21T00:00:00Z","timestamp":1692576000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2023,8,21]],"date-time":"2023-08-21T00:00:00Z","timestamp":1692576000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100010664","name":"H2020 Future and Emerging Technologies","doi-asserted-by":"publisher","award":["899417"],"award-info":[{"award-number":["899417"]}],"id":[{"id":"10.13039\/100010664","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100012774","name":"Innovationsfonden","doi-asserted-by":"publisher","award":["8088\u201000049B"],"award-info":[{"award-number":["8088\u201000049B"]}],"id":[{"id":"10.13039\/100012774","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Cancer Medicine"],"published-print":{"date-parts":[[2023,9]]},"abstract":"Abstract<\/jats:title>\n \n Backgrounds<\/jats:title>\n Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cell\u2010free DNA in human plasma.<\/jats:p>\n <\/jats:sec>\n \n Aims<\/jats:title>\n First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsy\u2010based assays.<\/jats:p>\n <\/jats:sec>\n \n Materials & Methods<\/jats:title>\n For the comparison we tested a solid\u2010phase silica purification method and a phenol\/chloroform method with salt precipitation.<\/jats:p>\n \n For the diagnostic application of eccDNA we developed and tested a qPCR primer\u2010based SNP detection system, for the detection of two well\u2010established cancer\u2010causing\n KRAS<\/jats:italic>\n mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing\n KRAS<\/jats:italic>\n mutant alleles in 0.5\u2009mL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls.\n <\/jats:p>\n <\/jats:sec>\n \n Results<\/jats:title>\n In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol\/chloroform method (15.7%\u201326.7%) compared with the solid\u2010phase purification approach (47.8%\u201365.9%).<\/jats:p>\n \n For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10\n \u22123<\/jats:sup>\n in a background of 10\n 5<\/jats:sup>\n wild type (wt)\n KRAS<\/jats:italic>\n circular entities, which was not improved by general amplification or primer\u2010based inhibition of wt\n KRAS<\/jats:italic>\n amplification. Furthermore, we did not detect eccDNA containing\n KRAS<\/jats:italic>\n in any of the clinical samples.\n <\/jats:p>\n <\/jats:sec>\n \n Discussion<\/jats:title>\n \n A potential explanation for our inability to detect any\n KRAS<\/jats:italic>\n mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma.\n <\/jats:p>\n <\/jats:sec>\n \n Conclusion<\/jats:title>\n Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primer\u2010based qPCR detection.<\/jats:p>\n <\/jats:sec>","DOI":"10.1002\/cam4.6385","type":"journal-article","created":{"date-parts":[[2023,8,21]],"date-time":"2023-08-21T22:53:11Z","timestamp":1692658391000},"page":"17679-17691","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Methods for the purification and detection of single nucleotide\n \n KRAS<\/i>\n <\/scp>\n mutations on extrachromosomal circular\n DNA<\/scp>\n in human plasma"],"prefix":"10.1002","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7523-2306","authenticated-orcid":false,"given":"Lasse","family":"B\u00f8llehuus\u00a0Hansen","sequence":"first","affiliation":[{"name":"Department of Biology University of Copenhagen Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra Fugl","family":"Jakobsen","sequence":"additional","affiliation":[{"name":"Department of Biology University of Copenhagen Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Egija","family":"Zole","sequence":"additional","affiliation":[{"name":"Department of Biology University of Copenhagen Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julie Boertmann","family":"Noer","sequence":"additional","affiliation":[{"name":"Department of Biology University of Copenhagen Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li Tai","family":"Fang","sequence":"additional","affiliation":[{"name":"Roche Sequencing Solutions Belmont California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sefa","family":"Alizadeh","sequence":"additional","affiliation":[{"name":"Department of Biology University of Copenhagen Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia Sidenius","family":"Johansen","sequence":"additional","affiliation":[{"name":"Department of Oncology Copenhagen University Hospital Herlev Denmark"},{"name":"Department of Medicine Copenhagen University Hospital Herlev Denmark"},{"name":"Department of Clinical Medicine, Faculty of Health and Medical Sciences University of Copenhagen Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marghoob","family":"Mohiyuddin","sequence":"additional","affiliation":[{"name":"Roche Sequencing Solutions Belmont California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Birgitte","family":"Regenberg","sequence":"additional","affiliation":[{"name":"Department of Biology University of Copenhagen Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,8,21]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrdp.2016.22"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.3322\/caac.21708"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.18632\/oncotarget.11061"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molonc.2016.01.007"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms21207651"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.trsl.2018.07.008"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1101\/gr.275691.121"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-1272-6"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12967-018-1677-2"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.5301\/jbm.5000088"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1914949117"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1158\/1541-7786.MCR-17-0095"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.onc.1200917"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mehy.2018.02.018"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/0921-8777(93)90071-n"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.7150\/ijbs.54614"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.2174\/1566524011313030003"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(88)90571-5"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ejca.2015.11.012"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/16.16.7773"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature11547"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1164368"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.21037\/tcr.2018.10.19"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-47489-7"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-319-42044-8_3"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.3007094"},{"key":"e_1_2_9_28_1","doi-asserted-by":"publisher","DOI":"10.3390\/diagnostics11040650"},{"issue":"8","key":"e_1_2_9_29_1","first-page":"232","article-title":"Identification 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