Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi

Autor(es)

da Silva, Marcelo S. ^{[1, 2]} ; Marini, Paula A. ^[2] ; Repoles, Bruno M. ^[3] ; Elias, Maria C. ^[2] ; Machado, Carlos R. ^[3]

Afiliação do(s) autor(es)

^[1] Univ Glasgow, Inst Infect Immun & Inflammat, Wellcome Ctr Mol Parasitol, Glasgow, Lanark - Scotland

^[2] Butantan Inst, Ctr Toxins Immune Response & Cell Signaling CeTIC, LECC, Sao Paulo - Brazil

^[3] Univ Fed Minas Gerais, Dept Bioquim & Imunol, Belo Horizonte, MG - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BIO-PROTOCOL; v. 8, n. 24 DEC 20 2018.
• Citações Web of Science: 0

Resumo

Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5'-chloro-2'deoxyuridine (CldU) and 5'-iodo-2'-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection. (AU)

• Processo FAPESP: 13/07467-1 - CeTICS - Centro de Toxinas, Imuno-Resposta e Sinalização Celular
• Beneficiário: Hugo Aguirre Armelin
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 14/24170-5 - Dinâmica da replicação do DNA em Trypanosoma cruzi: caracterização do licenciamento e da taxa de replicação
• Beneficiário: Marcelo Santos da Silva
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 17/18719-2 - Análise estrutural do complexo de reconhecimento de origens de replicação (ORC) em Trypanosoma brucei utilizando cryo-electron microscopy e single-particle analysis
• Beneficiário: Marcelo Santos da Silva
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Pós-Doutorado