Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture
Mem. Inst. Oswaldo Cruz
;
109(4): 511-513, 03/07/2014. graf
Artigo
em Inglês
| LILACS
| ID: lil-716313
ABSTRACT
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.
Texto completo:
DisponíveL
Índice:
LILACS (Américas)
Assunto principal:
Trypanosoma cruzi
Tipo de estudo:
Estudo diagnóstico
Idioma:
Inglês
Revista:
Mem. Inst. Oswaldo Cruz
Assunto da revista:
Medicina Tropical
/
Parasitologia
Ano de publicação:
2014
Tipo de documento:
Artigo
País de afiliação:
Brasil
Instituição/País de afiliação:
Instituto Carlos Chagas-Fiocruz/BR
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