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A non-destructive enzymatic method to extract DNA from arthropod specimens: Implications for morphological and molecular studies.

ABSTRACT: There is a growing necessity to integrate morphological and genetic studies. This paper proposes a new technique that allows DNA extraction of arthropods while still keeping intact the entire morphology of the specimens. The technique uses Proteinase K to dissolve protein tissues and preserve the chitinous exoskeleton of specimens. The method is fast, cheap, non-toxic, and allows for good morphological preparations of specimens retaining much of their tridimensional structure. The methodology works fine with specimens preserved in different kinds of media, such as for dry (pinned) specimens, and specimens preserved in Ethanol. In addition, it allows the extraction of DNA from fresh specimens, as well as from specimens preserved for a long time. The technique works well for morphological studies alone, but allows the generation of an associated genomic library at an individual-scale. Among the advantages of the new technique is the possibility of extracting DNA from the entire specimen (necessary for the study of diseases transmitted by arthropod vectors), while still keeping the morphology intact for correct taxonomic identification. In addition, in comparison with methods that extract DNA from small tissue samples (e.g., from legs or wings), the method allows for the extraction of a larger amount of DNA and is better suited for small specimens.

SUBMITTER: Santos D

PROVIDER: S-EPMC5794151 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A non-destructive enzymatic method to extract DNA from arthropod specimens: Implications for morphological and molecular studies.

Santos Daubian D Ribeiro Guilherme Cunha GC Cabral Aline Diniz AD Sperança Márcia Aparecida MA

PloS one 20180201 2

There is a growing necessity to integrate morphological and genetic studies. This paper proposes a new technique that allows DNA extraction of arthropods while still keeping intact the entire morphology of the specimens. The technique uses Proteinase K to dissolve protein tissues and preserve the chitinous exoskeleton of specimens. The method is fast, cheap, non-toxic, and allows for good morphological preparations of specimens retaining much of their tridimensional structure. The methodology wo ...[more]

PMID: 29390036

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Project description:Background and Objectives: The gold standard for a successful prosthetic approach is the osseointegration of an implant. However, this integration can be a problem in cases where the implant needs to be removed. Removing the implant with minimal damage to the surrounding tissues is important. Osteocytes cannot survive below -2 °C, but epithelial cells, fibroblasts, and other surrounding tissue cells can. Remodeling can be triggered by cryotherapy at temperatures that specifically affect osteocyte necrosis. In this study, we aimed to develop a method for reversing the osseointegration mechanism and for protecting the surrounding tissues by bone remodeling induced by CO2 cryotherapy. Materials and Methods: In this study, eight 2.8 mm diameter, one-piece mini implants were used in New Zealand rabbit tibias. Two control and six implants were tested in this study. After 2 months of osseointegration, a reverse torque force method was used to remove all osseointegrated implants at 5, 10, 20, and 30 Ncm. The osseointegration of the implants was proven by periotest measurements. Changes in bone tissue were examined in histological sections stained with toluidine blue after rabbit sacrifice. The number of lacunae with osteocyte, empty lacunae, and lacunae greater than 5 µm and the osteon number in a 10,000 µm2 area were calculated. Cryotherapy was applied to the test implants for 1 min, 2 min, and 5 min. Three implants were subjected to cryotherapy at -40 °C, and the other implants were subjected to cryotherapy at -80 °C. Results: Empty lacunae, filled osteocytes, lacunae >5 µm, and the osteon count around the implant applied at -40 °C were not significantly different from the control implants. The application of -40 °C for 1 min was found to cause minimal damage to the bone cells. The implants, which were applied for 1 min and 2 min, were successfully explanted on the 2nd day with the 5 Ncm reverse torque method. Test implants, which were applied cold for 5 min, were explanted on day 1. Tissue damage was detected in all test groups at -80 °C. Conclusions: The method of removing implants with cryotherapy was found to be successful in -40 °C freeze-thaw cycles applied three times for 1 min. To prove implant removal with cryotherapy, more implant trials should be conducted.

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A non-destructive enzymatic method to extract DNA from arthropod specimens: Implications for morphological and molecular studies.

Publications

A non-destructive enzymatic method to extract DNA from arthropod specimens: Implications for morphological and molecular studies.

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