Comparison of Different DNA Isolation Protocols in Old and New Insect Samples

Abstract

Insects belong to the Arthropoda phylum and are the most numerous animal group in the world, with more than 1.000.000 species. Insects ensure the sustainability of life on earth by pollinating plants, improving soil texture by decomposing organic materials, and providing nutrients to humans. DNA isolation is a necessity for performing analyses such as Sanger sequencing, DNA fingerprinting, and gene-genome analysis. Isolated insect DNA samples can be used to carry out studies such as species identification, taxonomic relations, comparison of insect-plant and –microfauna relations, revealing genomic, population, and genetic diversity, bio-geography determination, combating agricultural pests, etc. In this study, it is aimed to compare three different isolation methods in terms of time and quality for obtaining genetic material from museum samples (old) and new samples of insects. The samples used in this investigation belong to the same insect species of Cerambycidae family (Coleoptera). Samples collected at most 9-12 months ago are considered as “new specimen”, whereas “old specimens” consist samples of 7-28 years old. Three DNA isolation protocols: Phenol-Chloroform, Salting Out and DArT seq, are compared in this study. Considering quality gel images and A260/280 and A260/230 values in newly collected samples, the best results in terms of quality were obtained with the phenol-chloroform method. When A260/280 values were examined alone, DArT seq protocol appears to be the most appropriate protocol in terms of purity in both old and new insect samples. In terms of time, the Phenol-chloroform method was found to be the shortest protocol lasting for about 2 h. Accordingly, the phenol-chloroform method was deduced to be the better choice for both old and new insect samples. After concentration adjustments, the obtained DNAs can be used for the next experimental stages or stored as stock material at -20 ºC. Determining the appropriate protocol to obtain high-quality DNA in a short time is important for the continuation of the studies. Our findings are important in terms of accelerating the studies for identification and determining systematic status and especially in examining the taxonomic relationships of the insect species. Using molecular taxonomy in addition to classical taxonomy allows the identification of insects on a species basis. These types of studies will generate useful information for researchers who study insect molecular biology.