PCR Screening of Toxoplasma gondii Single Clones Directly from 96-Well Plates Without DNA Purification
Toxoplasma gondii has become a model for studying the phylum Apicomplexa, and more in general parasite-host interactions, thanks to its ease of growth in culture and availability of a broad array of genetics tools. Assigning gene function typically involves genetic techniques such as gene knockout, conditional expression, or protein tagging. These approaches generally require isolation of single clones that have correctly introduced the desired genetic modification into the target genomic locus. The frequency of positive clones carrying these genetic manipulations depends on the particular parasite strain and the impact that these genome modifications have on parasite fitness. An adverse effect on parasite viability or growth would result in a low abundancy of the correct transgenic parasites within the transfected population. This in turn will account for a low rate of positive clones after population cloning, requiring the genetic analysis of a high number of single clones. We have developed a simple and fast method to screen single clones of T. gondii directly from the 96-well plates without previous parasite expansion or time-consuming genomic extraction. This approach permits screening at an earlier point than previously possible, thus allowing for faster movement toward assessing gene function.
Key wordsToxoplasma gondii Parasite cloning PCR screening Phire Hot Start II DNA Polymerase
Financial Support: This work was supported by the U.S. National Institutes of Health (V.B.C. and M.D.C., grant number R01AI120607) and DCBB, University of Perugia, Italy, project FRB_2014.
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