Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture
Autor(a) principal: | |
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Data de Publicação: | 2014 |
Outros Autores: | , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da FIOCRUZ (ARCA) |
Texto Completo: | https://www.arca.fiocruz.br/handle/icict/16090 |
Resumo: | CNPq, FIOCRUZ |
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repository_id_str |
2135 |
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Alcantara, Monica VisnieskiFragoso, Stenio PerdigãoPicchi, Gisele Fernanda Assine2016-10-05T14:59:09Z2016-10-05T14:59:09Z2014ALCANTARA, Monica Visnieski; FRAGOSO, Stenio Perdigão; PICCHI, Gisele Fernanda Assine. Memórias do Instituto Oswaldo Cruz, Rio de Janeiro, v. 109, n. 4, p. 511-513, June 2014.0074-0276https://www.arca.fiocruz.br/handle/icict/1609010.1590/0074-0276140010CNPq, FIOCRUZFundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Molecular de Tripanossomatídeos. Curitiba, PR, Brasil / Universidade Federal do Paraná. Departamento de Biologia Celular e Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Molecular de Tripanossomatídeos. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Molecular de Tripanossomatídeos. Curitiba, PR, Brasil.Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106 -108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the “culture PCR” approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation.engClaude PirmezKnockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid cultureinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleCulture PCRKnockout confirmationTrypanosoma cruziColony PCRGenotype interrogationTransfectant cultureinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZLICENSElicense.txtlicense.txttext/plain; charset=utf-83074https://www.arca.fiocruz.br/bitstream/icict/16090/1/license.txt0271fb68963c017aa933c7aff60379f4MD51ORIGINALKnockout confirmation for Hurries.pdfKnockout confirmation for 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dc.title.pt_BR.fl_str_mv |
Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture |
title |
Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture |
spellingShingle |
Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture Alcantara, Monica Visnieski Culture PCR Knockout confirmation Trypanosoma cruzi Colony PCR Genotype interrogation Transfectant culture |
title_short |
Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture |
title_full |
Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture |
title_fullStr |
Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture |
title_full_unstemmed |
Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture |
title_sort |
Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture |
author |
Alcantara, Monica Visnieski |
author_facet |
Alcantara, Monica Visnieski Fragoso, Stenio Perdigão Picchi, Gisele Fernanda Assine |
author_role |
author |
author2 |
Fragoso, Stenio Perdigão Picchi, Gisele Fernanda Assine |
author2_role |
author author |
dc.contributor.author.fl_str_mv |
Alcantara, Monica Visnieski Fragoso, Stenio Perdigão Picchi, Gisele Fernanda Assine |
dc.subject.en.pt_BR.fl_str_mv |
Culture PCR Knockout confirmation Trypanosoma cruzi Colony PCR Genotype interrogation Transfectant culture |
topic |
Culture PCR Knockout confirmation Trypanosoma cruzi Colony PCR Genotype interrogation Transfectant culture |
description |
CNPq, FIOCRUZ |
publishDate |
2014 |
dc.date.issued.fl_str_mv |
2014 |
dc.date.accessioned.fl_str_mv |
2016-10-05T14:59:09Z |
dc.date.available.fl_str_mv |
2016-10-05T14:59:09Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
ALCANTARA, Monica Visnieski; FRAGOSO, Stenio Perdigão; PICCHI, Gisele Fernanda Assine. Memórias do Instituto Oswaldo Cruz, Rio de Janeiro, v. 109, n. 4, p. 511-513, June 2014. |
dc.identifier.uri.fl_str_mv |
https://www.arca.fiocruz.br/handle/icict/16090 |
dc.identifier.issn.pt_BR.fl_str_mv |
0074-0276 |
dc.identifier.doi.pt_BR.fl_str_mv |
10.1590/0074-0276140010 |
identifier_str_mv |
ALCANTARA, Monica Visnieski; FRAGOSO, Stenio Perdigão; PICCHI, Gisele Fernanda Assine. Memórias do Instituto Oswaldo Cruz, Rio de Janeiro, v. 109, n. 4, p. 511-513, June 2014. 0074-0276 10.1590/0074-0276140010 |
url |
https://www.arca.fiocruz.br/handle/icict/16090 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
Claude Pirmez |
publisher.none.fl_str_mv |
Claude Pirmez |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da FIOCRUZ (ARCA) instname:Fundação Oswaldo Cruz (FIOCRUZ) instacron:FIOCRUZ |
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Fundação Oswaldo Cruz (FIOCRUZ) |
instacron_str |
FIOCRUZ |
institution |
FIOCRUZ |
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Repositório Institucional da FIOCRUZ (ARCA) |
collection |
Repositório Institucional da FIOCRUZ (ARCA) |
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