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

Abstract

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.

Clinical Outcomes and Co-Occurring Mutations in Patients with RUNX1-Mutated Acute Myeloid Leukemia

“…Diagnostic bone marrow samples were obtained for mutational analysis. Total genomic DNA was extracted from unenriched peripheral blood (PB) or bone marrow (BM) samples using ReliaPrep genomic DNA isolation kit (Promegal Corp, Madison, WI, USA) [ 31 ]. FLT3 (internal tandem duplication and D835) was assessed by PCR followed by capillary electrophoresis on a Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) [ 32 ].…”

Open the SterivexTM casing: An easy and effective way to improve DNA extraction yields

“…After collecting samples, the method used to extract the DNA from filters then has to yield sufficient DNA for subsequent molecular analyses. Indeed, new sequencing technologies may require high concentrations of extracted DNA which could be difficult to reach for some types of samples (Bürgmann et al ; Casaril et al , Illumina manufacturer's instructions). However, it is unclear whether standard extraction methods are achieving the maximum possible yields of DNA from these encapsulated filter units.…”

>b<Aspectos bioecológicos de flebotomíneos (Diptera: Psychodidae: Phlebotominae) em área endêmica para leishmaniose visceral no estado de São Paulo>/b<

“…Atualmente, as análises moleculares mostram-se ferramentas poderosas nos estudos de bioecologia de flebotomíneos, apoiando a caracterização de vetores e não vetores, a identificação de espécies crípticas ou ainda, a proposição de novas sistemáticas evolutivas (Casaril et al, 2017;Depaquit, 2014). Dessa forma, uma quantidade suficiente de DNA de alta qualidade é essencial para a utilização dessas ferramentas e a seleção de um método apropriado de extração de DNA desempenha um papel central nesse sentido (Psifidi et al, 2010 (Casaril et al, 2017;Yang et al, 1998 Esses métodos foram utilizados em vários estudos moleculares com flebotomíneos Casaril et al, 2017;González et al, 2015;Sales et al, 2015 Paiva et al (2007; esse método não mostrou boa sensibilidade; além disso, também foi observada perda de DNA durante o processo de lavagens. Da mesma forma, Arez et al (2000) observaram uma possível inibição da PCR pela presença de inibidores no tecido do mosquito Anopheles spp.…”

“…Nos últimos anos, custo das análises moleculares caiu (Depaquit, 2014). Assim, as ferramentas moleculares começaram a ser incluídas nos estudos de flebotomíneos, aumentando a sensibilidade e a especificidade na detecção de DNA do parasita (Paiva et al, 2007) e/ou ajudando na identificação taxonômica desses dípteros (Casaril et al, 2017).…”

“…Como se sabe, para análises moleculares eficientes, é extremamente importante obter DNA livre de contaminantes e em quantidades suficientes (Oliveira et al, 2007) fato, que é de grande preocupação em amostras de flebotomíneos que possuem corpos pequenos constituídos, principalmente, por um exoesqueleto de quitina, que pode atuar como inibidor de PCR (Casaril et al, 2017;Paiva et al, 2007). Além disso, o processo de preservação desses insetos, após a captura, é uma importante etapa anterior que deve ser levada em consideração pelos pesquisadores, a afim de realizar análises moleculares mais sensíveis e específicas (Paiva et al, 2007 (Riemann et al, 2007).…”