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- Publisher Website: 10.1093/nar/gkn523
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- PMID: 18710883
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Article: Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs
Title | Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs |
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Authors | |
Issue Date | 2008 |
Citation | Nucleic Acids Research, 2008, v. 36, n. 18 How to Cite? |
Abstract | We evaluate here the use of real-time quantitative PCR (q-PCR) as a method for screening for homologous recombinants generated in mammalian cells from either conventional gene-targeting constructs or whole BAC-based constructs. Using gene-targeted events at different loci, we show that q-PCR is a highly sensitive and accurate method for screening for conventional gene targeting that can reduce the number of clones requiring follow-up screening by Southern blotting. We further compared q-PCR to fluorescent in situ hybridization (FISH) for the detection of gene-targeting events using full-length BAC-based constructs designed to introduce mutations either into one gene or simultaneously into two adjacent genes. We find that although BAC-based constructs appeared to have high rates of homologous recombination when evaluated by FISH, screening by FISH was prone to false positives that were detected by q-PCR. Our results demonstrate the utility of q-PCR as a screening tool for gene targeting and further highlight potential problems with the use of whole BAC-based constructs for homologous recombination. © 2008 The Author(s). |
Persistent Identifier | http://hdl.handle.net/10722/249027 |
ISSN | 2023 Impact Factor: 16.6 2023 SCImago Journal Rankings: 7.048 |
ISI Accession Number ID |
DC Field | Value | Language |
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dc.contributor.author | Gómez-rodrÃguez, Julio | - |
dc.contributor.author | Washington, Valance | - |
dc.contributor.author | Cheng, Jun | - |
dc.contributor.author | Dutra, Amalia | - |
dc.contributor.author | Pak, Evgenia | - |
dc.contributor.author | Liu, Pentao | - |
dc.contributor.author | Mcvicar, Daniel W. | - |
dc.contributor.author | Schwartzberg, Pamela L. | - |
dc.date.accessioned | 2017-10-27T05:58:54Z | - |
dc.date.available | 2017-10-27T05:58:54Z | - |
dc.date.issued | 2008 | - |
dc.identifier.citation | Nucleic Acids Research, 2008, v. 36, n. 18 | - |
dc.identifier.issn | 0305-1048 | - |
dc.identifier.uri | http://hdl.handle.net/10722/249027 | - |
dc.description.abstract | We evaluate here the use of real-time quantitative PCR (q-PCR) as a method for screening for homologous recombinants generated in mammalian cells from either conventional gene-targeting constructs or whole BAC-based constructs. Using gene-targeted events at different loci, we show that q-PCR is a highly sensitive and accurate method for screening for conventional gene targeting that can reduce the number of clones requiring follow-up screening by Southern blotting. We further compared q-PCR to fluorescent in situ hybridization (FISH) for the detection of gene-targeting events using full-length BAC-based constructs designed to introduce mutations either into one gene or simultaneously into two adjacent genes. We find that although BAC-based constructs appeared to have high rates of homologous recombination when evaluated by FISH, screening by FISH was prone to false positives that were detected by q-PCR. Our results demonstrate the utility of q-PCR as a screening tool for gene targeting and further highlight potential problems with the use of whole BAC-based constructs for homologous recombination. © 2008 The Author(s). | - |
dc.language | eng | - |
dc.relation.ispartof | Nucleic Acids Research | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.title | Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs | - |
dc.type | Article | - |
dc.description.nature | published_or_final_version | - |
dc.identifier.doi | 10.1093/nar/gkn523 | - |
dc.identifier.pmid | 18710883 | - |
dc.identifier.scopus | eid_2-s2.0-54549120496 | - |
dc.identifier.volume | 36 | - |
dc.identifier.issue | 18 | - |
dc.identifier.spage | null | - |
dc.identifier.epage | null | - |
dc.identifier.eissn | 1362-4962 | - |
dc.identifier.isi | WOS:000260152900031 | - |
dc.identifier.issnl | 0305-1048 | - |