Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture

Detalhes bibliográficos
Autor(a) principal: Alcantara, Monica Visnieski
Data de Publicação: 2014
Outros Autores: Fragoso, Stenio Perdigão, Picchi, Gisele Fernanda Assine
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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spelling 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
instname_str Fundação Oswaldo Cruz (FIOCRUZ)
instacron_str FIOCRUZ
institution FIOCRUZ
reponame_str Repositório Institucional da FIOCRUZ (ARCA)
collection Repositório Institucional da FIOCRUZ (ARCA)
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