BR-102017026429-B1 - METHOD FOR SUBSEQUENT EXTRACTION OF FATTY ACIDS AND GENOMIC DNA FROM DIPTERA

BR102017026429B1BR 102017026429 B1BR102017026429 B1BR 102017026429B1BR-102017026429-B1

Abstract

The present invention relates to a method for the subsequent extraction of fatty acids and total genomic DNA from dipterans of forensic importance. The process involves at least the following steps: immersion of samples in petroleum ether and dichloromethane for fatty acid extraction, followed by extraction of total genomic DNA using a silica gel membrane substrate from a column supplied in a commercial kit. The present invention eliminates the use of phenol and chloroform for extraction, and suppresses an alcohol precipitation step, as well as maintaining the complete integrity of the individuals used in the extraction process. The extracted samples can then be applied in biochemical and molecular biology methods, such as chromatography, polymerase chain reaction (PCR), and DNA sequencing. Since the identification of the developmental stage of dipterans is of great importance for forensic entomology, the present invention relates to an innovative process with the potential to be commercialized by forensic research and biotechnology laboratories.

Inventors

Vinicius Farias Campos
EDUARDO BIERHALS BLODORN
KATHLEEN TAVARES WINKEL
LETÍCIA BRAATZ FERREIRA
CLAUDIO MARTIN PEREIRA DE PEREIRA
LUCAS MORAES BERNEIRA
PATRICIA JACQUELINE THYSSEN
WILLIAM BORGES DOMINGUES

Assignees

UNIVERSIDADE FEDERAL DE PELOTAS

Dates

Publication Date: 20260310
Application Date: 20171207

Claims (3)

1. Subsequent extraction process of fatty acids and total genomic DNA from intact and individualized dipterans, characterized by comprising the following sequential steps: incubating at least one intact and individualized dipteran individual in petroleum ether, at room temperature, for a period of 48 hours, promoting the extraction of cuticular fatty acids; transferring the individual resulting from the first step to dichloromethane, maintaining incubation at room temperature, in order to complete the extraction of fatty acids; after the extraction of fatty acids, subjecting the individual to washes with an alcoholic solution, preferably 70% alcohol, aiming at the removal of organic residues; performing cell lysis of the individual by incubation in cell lysis buffer containing a proteolytic agent, preferably Proteinase K, at a temperature of approximately 56 °C, for a period between 24 and 36 hours, obtaining a cell lysate; Add a buffer containing a chaotropic agent to the cell lysate, followed by the addition of an alcoholic solution, promoting suitable conditions for the binding of genomic DNA to a solid substrate; purify the genomic DNA by binding to a silica gel membrane arranged in a column, followed by successive washing steps; and elute the purified genomic DNA, obtaining total genomic DNA suitable for applications in biochemical and molecular analyses, this process eliminating the need for phenol and chloroform and preserving the morphological integrity of the dipteran individual after the extraction steps.
2. Process according to claim 1, characterized in that the cell lysis step is performed using ATL buffer and Proteinase K, with incubation at approximately 56 °C, and in that the purification of genomic DNA is performed using a silica gel membrane column provided in a commercial DNA extraction kit.
3. A process according to any one of claims 1 or 2, characterized in that the genomic DNA obtained is applicable to selected molecular biology techniques including polymerase chain reaction (PCR), real-time PCR, DNA sequencing, and associated genomic analyses.

Description

Descriptive Report FIELD OF THE INVENTION [001] The present invention relates to a method for extracting fatty acids and total genomic DNA subsequently from individual diptera of forensic importance, using smaller volumes than those described and required in the scientific literature to date. More particularly, it relates to the protocol for obtaining fatty acids using petroleum ether and dichloromethane, and subsequent extraction of total genomic DNA. [002] In particular, the present invention relates to a process involving the immersion of insect samples of the order Diptera in petroleum ether, followed by their transfer to a dichloromethane solution at room temperature. After incubation in dichloromethane, the samples can be used individually to obtain fatty acids and subsequent extraction of total genomic DNA, using a silica gel membrane substrate from a column supplied in a commercial kit. [003] The present method for the subsequent extraction of fatty acids and total genomic DNA from diptera can be applied to individual samples with a simple and safe protocol, resulting in the obtaining of satisfactory quantities of fatty acids and DNA free of biological contaminants and interfering reagents. The present method also maintains the integrity of the samples, allowing the morphology of the insects to be reassessed after the extraction processes. The identification of diptera is of great importance for forensic entomology, therefore the present invention relates to an innovative process with the potential to be commercialized by forensic investigation and technological development laboratories. FUNDAMENTALS OF THE INVENTION Of the Diptera of forensic importance [004] Chrysomya species exhibit great ecological diversity. Chrysomya megacephala is an exotic species, introduced to Brazil along with Chrysomya putoria and Chrysomya albiceps, with its first record in Rio Grande do Sul. C. megacephala has a worldwide distribution and is reported as one of the predominant scavengers in carcass colonization. Immature stages develop on discrete and ephemeral substrates, represented by decomposing organic matter such as feces, carcasses, and viscera. Because it is among the first and predominant insects involved in cadaveric decomposition, it can be considered as evidence in the criminal field. [005] These insects are of great interest to forensic entomology. The immature stages, in particular, can help in estimating the post-mortem interval, as a biological sample for toxicological analysis, and also for DNA extraction, assisting in the identification of victims and/or suspects in cases of sexual crimes, since they feed directly on the corpse. [006] Proper insect identification is the most important element in the field of forensic entomology. It is species identification that allows for the appropriate use of developmental and distributional data. In this context, chemotaxonomy can bring some advances to facilitate the work of investigators, as it can differentiate insect orders through fatty acid profiles, since lipid composition can be intrinsic to species. Of the methods of fatty acid extraction [007] The qualitative and quantitative identification of fatty acids in biological samples is initially characterized by the extraction of these compounds. Extraction can be performed using conventional methods based on a mixture of methanol and chloroform; however, the sample needs to be lyophilized and ground. Recently, another methodology that has been widely used in insects is based on the use of petroleum ether and dichloromethane, which eliminates the need for lyophilization and grinding of the material, and requires less manipulation of the biological sample. [008] To date, petroleum ether and dichloromethane extraction has, above all, the great advantage of keeping the sample intact, and can also be used for subsequent taxonomic and molecular analyses. Genomic DNA extraction methods [009] Most conventional genomic DNA extraction methods require that all or part of the specimen be vigorously ground until no solid material remains apparent. The processes are generally followed by digestion of cell membranes with proteases, and subsequent use of chloroform, alcohol, phenol, among other organic solvents, to remove proteins and increase the retention of nucleic acids in silica gel membranes. Only after these steps is the genomic DNA separated and obtained in satisfactory quantity and quality. [010] Although efficient, conventional methods are laborious and still have some disadvantages, such as those listed below. The destruction of specimens can lead to the loss of important information, preventing morphological assessments from being challenged or reviewed. The use of organic solvents, such as chloroform and phenols, can not only harm the operator but also pollute the environment. Finally, sample treatment can impair the interaction of the extraction with other molecular tests, thus reducing the accuracy of the final