Standardization of DNA extraction from sand flies: Application to genotyping by next generation sequencing

Standardization of the methods for extraction of DNA from sand flies is essential for obtaining high efficiency during subsequent molecular analyses, such as the new sequencing methods. Information obtained using these methods may contribute substantially to taxonomic, evolutionary, and eco-epidemio...

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Bibliographic Details
Published in:	Experimental parasitology Vol. 177; pp. 66 - 72
Main Authors:	Casaril, Aline Etelvina, de Oliveira, Liliane Prado, Alonso, Diego Peres, de Oliveira, Everton Falcão, Gomes Barrios, Suellem Petilim, de Oliveira Moura Infran, Jucelei, Fernandes, Wagner de Souza, Oshiro, Elisa Teruya, Ferreira, Alda Maria Teixeira, Ribolla, Paulo Eduardo Martins, de Oliveira, Alessandra Gutierrez
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-06-2017
Subjects:	Animals Chi-Square Distribution Chloroform DNA - chemistry DNA - isolation & purification Endopeptidase K - metabolism Genotyping Techniques - methods Genotyping Techniques - standards Male Molecular biology Phenol Polymerase Chain Reaction - methods Psychodidae - genetics Sequence Analysis, DNA - methods Sequence Analysis, DNA - standards Statistics, Nonparametric Vector Visceral leishmaniasis Molecular biology Visceral leishmaniasis Vector
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Summary:	Standardization of the methods for extraction of DNA from sand flies is essential for obtaining high efficiency during subsequent molecular analyses, such as the new sequencing methods. Information obtained using these methods may contribute substantially to taxonomic, evolutionary, and eco-epidemiological studies. The aim of the present study was to standardize and compare two methods for the extraction of genomic DNA from sand flies for obtaining DNA in sufficient quantities for next-generation sequencing. Sand flies were collected from the municipalities of Campo Grande, Camapuã, Corumbá and Miranda, state of Mato Grosso do Sul, Brazil. Three protocols using a silica column-based commercial kit (ReliaPrep™ Blood gDNA Miniprep System kit, Promega®), and three protocols based on the classical phenol-chloroform extraction method (Uliana et al., 1991), were compared with respect to the yield and quality of the extracted DNA. DNA was quantified using a Qubit 2.0 fluorometer. The presence of sand fly DNA was confirmed by PCR amplification of the IVS6 region (constitutive gene), followed by electrophoresis on a 1.5% agarose gel. A total of 144 male specimens were analyzed, 72 per method. Significant differences were observed between the two methods tested. Protocols 2 and 3 of phenol-chloroform extraction presented significantly better performance than all commercial kit extraction protocols tested. For phenol-chloroform extraction, protocol 3 presented significantly better performance than protocols 1 and 2. The IVS6 region was detected in 70 of 72 (97.22%) samples extracted with phenol, including all samples for protocols 2 and 3. This is the first study on the standardization of methods for the extraction of DNA from sand flies for application to next-generation sequencing, which is a promising tool for entomological and molecular studies of sand flies. [Display omitted] •Three protocols developed for the commercial kit were found to be more practical.•Phenol/chloroform protocols resulted in significantly higher quantities of sand fly gDNA.•Higher quantities of proteinase K resulted in higher concentrations of extracted sand fly DNA.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0014-4894 1090-2449
DOI:	10.1016/j.exppara.2017.04.010