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{"title":"用核酸外切酶V-qPCR分析人乳头瘤病毒16在细胞系和组织中的基因组状态","authors":"Julia E. Myers, Katarzyna Zwolinska, Martin J. Sapp, Rona S. Scott","doi":"10.1002/cpmc.119","DOIUrl":null,"url":null,"abstract":"<p>Integration of the human papillomavirus (HPV) genome into host cell chromosomes has been observed in a majority of HPV-positive cervical cancers and a subset of oral HPV-associated cancers. HPV integration also occurs in long-term cell culture. Screening for HPV integration can be labor intensive and yield results that are difficult to interpret. Here we describe an assay based on exonuclease V (ExoV/RecBCD) and quantitative polymerase chain reaction (qPCR) to determine if samples from cell lines and tissues contain episomal or integrated HPV. This assay can be applied to screen other small DNA viruses with episomal/linear genome configurations in their viral lifecycle and has the potential to be used in clinical settings to define viral genomic conformations associated with disease. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Exonuclease V genomic DNA digestion and qPCR for detection of HPV16 genome configuration in cells</p><p><b>Support Protocol</b>: Exonuclease V analysis of HPV16 genome configuration in tissues</p><p><b>Alternate Protocol</b>: Determining HPV integration type or integrity of HPV episome</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2020-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.119","citationCount":"8","resultStr":"{\"title\":\"An Exonuclease V–qPCR Assay to Analyze the State of the Human Papillomavirus 16 Genome in Cell Lines and Tissues\",\"authors\":\"Julia E. Myers, Katarzyna Zwolinska, Martin J. Sapp, Rona S. Scott\",\"doi\":\"10.1002/cpmc.119\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Integration of the human papillomavirus (HPV) genome into host cell chromosomes has been observed in a majority of HPV-positive cervical cancers and a subset of oral HPV-associated cancers. HPV integration also occurs in long-term cell culture. Screening for HPV integration can be labor intensive and yield results that are difficult to interpret. Here we describe an assay based on exonuclease V (ExoV/RecBCD) and quantitative polymerase chain reaction (qPCR) to determine if samples from cell lines and tissues contain episomal or integrated HPV. This assay can be applied to screen other small DNA viruses with episomal/linear genome configurations in their viral lifecycle and has the potential to be used in clinical settings to define viral genomic conformations associated with disease. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Exonuclease V genomic DNA digestion and qPCR for detection of HPV16 genome configuration in cells</p><p><b>Support Protocol</b>: Exonuclease V analysis of HPV16 genome configuration in tissues</p><p><b>Alternate Protocol</b>: Determining HPV integration type or integrity of HPV episome</p>\",\"PeriodicalId\":39967,\"journal\":{\"name\":\"Current Protocols in Microbiology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2020-10-16\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1002/cpmc.119\",\"citationCount\":\"8\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current Protocols in Microbiology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cpmc.119\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Microbiology","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpmc.119","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
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