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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
da Silva, Marcelo S. [1, 2] ; Marini, Paula A. [2] ; Repoles, Bruno M. [3] ; Elias, Maria C. [2] ; Machado, Carlos R. [3]
Total Authors: 5
Affiliation:
[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
Total Affiliations: 3
Document type: Journal article
Source: BIO-PROTOCOL; v. 8, n. 24 DEC 20 2018.
Web of Science Citations: 0
Abstract

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)

FAPESP's process: 13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling
Grantee:Hugo Aguirre Armelin
Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
FAPESP's process: 14/24170-5 - DNA replication dynamics in Trypanosoma cruzi: licensing and replication rate characterization
Grantee:Marcelo Santos da Silva
Support Opportunities: Scholarships in Brazil - Post-Doctoral
FAPESP's process: 17/18719-2 - Structural analysis of the origin recognition complex (ORC) in Trypanosoma brucei using cryo-electron microscopy and single-particle analysis
Grantee:Marcelo Santos da Silva
Support Opportunities: Scholarships abroad - Research Internship - Post-doctor