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

Alcantara MV, Fragoso SP, Picchi GF - Mem. Inst. Oswaldo Cruz (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Molecular de Tripanossomatídeos, Instituto Carlos Chagas-Fiocruz, Curitiba, PR, Brasil.

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.

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: confirming knockout cassette insertion at the rightlocus by real-time polymerase chain reaction (PCR).Graphic representation of cycle threshold (Ct) number obtained afterreal-time PCR reaction allowing genotype comparison since all tested sampleswere obtained from roughly 2 x 107 cells/mL cultures. As expected,amplification of 40S ribosomal protein S4 (S4 control), triosephosphateisomerase (TI control) and histone H2AZ (H2AZ control) genes was about thesame in wild type (WT) and all seven different transformantTrypanosoma cruzi cultures. Hygromycin knockoutcassette was inserted at the right locus since samples fromtransformant population cultures were positives when amplified using aforward primer internal to the construction (IR2intF) and a reverse primerexternal to the cassette insertion (extR). No template control (data notshown) was included as negative control. Real-time PCR reactions wereperformed according standard protocols using 2 µL of DNA sample obtained asdescribed earlier.
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f03: : confirming knockout cassette insertion at the rightlocus by real-time polymerase chain reaction (PCR).Graphic representation of cycle threshold (Ct) number obtained afterreal-time PCR reaction allowing genotype comparison since all tested sampleswere obtained from roughly 2 x 107 cells/mL cultures. As expected,amplification of 40S ribosomal protein S4 (S4 control), triosephosphateisomerase (TI control) and histone H2AZ (H2AZ control) genes was about thesame in wild type (WT) and all seven different transformantTrypanosoma cruzi cultures. Hygromycin knockoutcassette was inserted at the right locus since samples fromtransformant population cultures were positives when amplified using aforward primer internal to the construction (IR2intF) and a reverse primerexternal to the cassette insertion (extR). No template control (data notshown) was included as negative control. Real-time PCR reactions wereperformed according standard protocols using 2 µL of DNA sample obtained asdescribed earlier.

Mentions: In the first test, we did not observe good amplification signals using samples preparedas if they were to be used for conventional PCR (data not shown). Assuming that thistest failed because PCR inhibitors are present in the liver infusion tryptose culturemedium and real-time PCR is more sensitive to them, we decided to evaluate whetherdilutions of the samples could reduce inhibitors, enabling correct amplifications.Indeed, compared with the results obtained for genomic DNA, we conclude that the samplesmust be diluted to avoid reaction inhibition by the culture medium components.Furthermore, we propose that a 1:200 dilution should be used for real-time PCR reactions(instead of the 1:1 dilution for conventional PCR) because it was the best result, witha smaller cycle threshold (Ct) deviation (Supplementary data). Thus,requiring no standard curve and using only the Ct information, we were able tocorroborate the use of real-time PCR results for the determination of knockout cassetteinsertion in the cultures of different transformants (Fig. 3).


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

Alcantara MV, Fragoso SP, Picchi GF - Mem. Inst. Oswaldo Cruz (2014)

: confirming knockout cassette insertion at the rightlocus by real-time polymerase chain reaction (PCR).Graphic representation of cycle threshold (Ct) number obtained afterreal-time PCR reaction allowing genotype comparison since all tested sampleswere obtained from roughly 2 x 107 cells/mL cultures. As expected,amplification of 40S ribosomal protein S4 (S4 control), triosephosphateisomerase (TI control) and histone H2AZ (H2AZ control) genes was about thesame in wild type (WT) and all seven different transformantTrypanosoma cruzi cultures. Hygromycin knockoutcassette was inserted at the right locus since samples fromtransformant population cultures were positives when amplified using aforward primer internal to the construction (IR2intF) and a reverse primerexternal to the cassette insertion (extR). No template control (data notshown) was included as negative control. Real-time PCR reactions wereperformed according standard protocols using 2 µL of DNA sample obtained asdescribed earlier.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4155859&req=5

f03: : confirming knockout cassette insertion at the rightlocus by real-time polymerase chain reaction (PCR).Graphic representation of cycle threshold (Ct) number obtained afterreal-time PCR reaction allowing genotype comparison since all tested sampleswere obtained from roughly 2 x 107 cells/mL cultures. As expected,amplification of 40S ribosomal protein S4 (S4 control), triosephosphateisomerase (TI control) and histone H2AZ (H2AZ control) genes was about thesame in wild type (WT) and all seven different transformantTrypanosoma cruzi cultures. Hygromycin knockoutcassette was inserted at the right locus since samples fromtransformant population cultures were positives when amplified using aforward primer internal to the construction (IR2intF) and a reverse primerexternal to the cassette insertion (extR). No template control (data notshown) was included as negative control. Real-time PCR reactions wereperformed according standard protocols using 2 µL of DNA sample obtained asdescribed earlier.
Mentions: In the first test, we did not observe good amplification signals using samples preparedas if they were to be used for conventional PCR (data not shown). Assuming that thistest failed because PCR inhibitors are present in the liver infusion tryptose culturemedium and real-time PCR is more sensitive to them, we decided to evaluate whetherdilutions of the samples could reduce inhibitors, enabling correct amplifications.Indeed, compared with the results obtained for genomic DNA, we conclude that the samplesmust be diluted to avoid reaction inhibition by the culture medium components.Furthermore, we propose that a 1:200 dilution should be used for real-time PCR reactions(instead of the 1:1 dilution for conventional PCR) because it was the best result, witha smaller cycle threshold (Ct) deviation (Supplementary data). Thus,requiring no standard curve and using only the Ct information, we were able tocorroborate the use of real-time PCR results for the determination of knockout cassetteinsertion in the cultures of different transformants (Fig. 3).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Molecular de Tripanossomatídeos, Instituto Carlos Chagas-Fiocruz, Curitiba, PR, Brasil.

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.

Show MeSH