Abstract
Trypanosome mitochondrial genome, known as Kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings. The studies of kDNA replication and architecture are of major significance since kDNA is a valid drug target. However, DNA in procyclic trypanosomes can not be labeled with tracer concentrations of 3[H]-thymidine, possibly because they lack a high-affinity transporter for thymidine. Therefore, BrdU, a thymidine analog, has been used at high concentrations to study kDNA replication. However, the detection of BrdU with anti-BrdU antibody requires harsh conditions such as the acid or heat treatment to seperate double DNA strand, which affects the ability for other antibodies to bind as well as the morphology and ability for dyes that require dsDNA to bind efficiently. Instead, EdU (5-Ethynyl-2′-deoxyuridine), a novel thymidine analog, can be used to study kDNA replication and cell proliferation with a simplified protocol. Detection of EdU is based on a click reaction, which is a copper (I) catalyzed reaction between an azide and an alkyne. This click reaction does not require DNA denaturation and it is multiplex compatible, such as other antibodies and dyes for cell cycle analyses. To visualize trypanosome replicating nuclear DNA and kDNA, EdU is added into the medium of cell culture and incubated for 0.5 h to 3 h and then detected by the following procedures.
Keywords: Trypanosome, Kinetoplast DNA, EdU labeling, KDNA, Trypanosoma brucei
Materials and Reagents
Equipment
Procedure
I. EdU labeling of trypanosome cells
Notes: 1) This protocol has not been used for the bloodstream form of trypanosomes yet. However, it is supposed to work well for the bloodstream form of trypanosomes too. 2) From step 3 to the end of the protocol, operate at room temperature (RT). 3) From step 9, please protect from light by covering the box with lid or the eppendorf tubes with foil paper. In steps 7, 8, and 10, use a 100 or 200 μl pipette to remove solution from each well.
II. EdU labeling of kDNA networks isolated from trypanosomes
Notes: 1) From step 3 to the end of the protocol, operate at RT (room temperature). 2) From step 7, please protect from light by covering the box with lid or the eppendorf tubes with foil paper. 3) In steps 8 and 9, please use a 100 or 200 μl pipette to remove solution from each well.
Recipes
Acknowledgments
I thank all lab members of Paul Englund and Robert Jensen for helpful discussions. This work was supported by NIH grant AI058613.
References
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