Comparative study on the effect of preservatives and extraction methods on the quality of spider DNA

Owing to recent innovation in molecular biological techniques, nucleic acid data are becoming more and more important in ecology, systematics, evolutionary biology and conservation biology. Moreover, DNA sequence comparison is a powerful tool to study genetic relatedness of species, which can be used to estimate branching order of phylogenetic trees as well as evolutionary distance between both extinct and extant taxa. To prepare an accurate phylogenetic tree it is necessary to use a preservative which causes less degradation of DNA. It is possible to obtain DNA from dried insects, but this is not an option for arachnids, which require wet preservation to facilitate identification using structures that shrivel when dried. In this context, the effect of three preservatives (acetone, ethanol (70%) and formalin (4%)) on DNA of the spider Pisaura mirabilis were tested and compared with fresh specimens in this study. For all treatments, spiders were placed directly in 1.5 ml preservative, kept in room temperature for 3 months and DNA was extracted from the leg of the preserved spider by four different DNA extraction methods (DNeasy method, Quick Extraction method, Master Pure method and CTAB method). PCR quality of the extracted DNA was tested with agarose gel electrophoresis. This study revealed that the quality of the extracted DNA varies and the variation seems related to the mode of preservation and extraction method. From the results it is evident that formalin, which is a routine preservative, has poor capacity to retain the integrity of DNA. Further, ethanol preservation degrades DNA to some extent but gives separation of the strands in extraction. Acetone did not affect the quality of DNA and the yield was comparable to that of fresh specimen. DNA shearing and inter-strand cross linking which occurred both in formalin and ethanol preserved specimens did not occur in acetone. By comparing the results, it can be concluded that acetone is superior to ethanol and formalin in preservation because it yields high quality DNA which can be compared to fresh samples in all the extraction methods tested. Formalin reacts with DNA through interaction with hydrogen bonds, fixation and denaturation of DNA proteins, cross linking between proteins and DNA and methylation of the nucleic acid. Acetone was found to be robust against water contamination than ethanol. Considering that most biological materials contain high amounts of water, acetone may be more recommended as preservative than ethanol which is widely used for the purpose. KEYWORDS