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

dc.creatorAlcantara, Monica Visnieski
dc.creatorFragoso, Stenio Perdigão
dc.creatorPicchi, Gisele Fernanda Assine
dc.date2016-10-05T14:59:09Z
dc.date2016-10-05T14:59:09Z
dc.date2014
dc.date.accessioned2023-09-26T20:41:40Z
dc.date.available2023-09-26T20:41:40Z
dc.identifierALCANTARA, 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.identifier0074-0276
dc.identifierhttps://www.arca.fiocruz.br/handle/icict/16090
dc.identifier10.1590/0074-0276140010
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8862146
dc.descriptionCNPq, FIOCRUZ
dc.descriptionGene 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.
dc.formatapplication/pdf
dc.languageeng
dc.publisherClaude Pirmez
dc.rightsopen access
dc.subjectCulture PCR
dc.subjectKnockout confirmation
dc.subjectTrypanosoma cruzi
dc.subjectColony PCR
dc.subjectGenotype interrogation
dc.subjectTransfectant culture
dc.titleKnockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture
dc.typeArticle


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