Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Abstract

word count: 198. Abstract 27 28 The principle and techniques applied in DNA extraction play a pivotal role in the obtention 29 of a considerable and purified yield of genetic material. In this work, we evaluate the 30 efficiency of seven protocols chosen from literature for the DNA extraction of Hypostomus 31 commersoni (Valenciennes, 1836), an important component of South American freshwater 32 ichthyofauna. We also proposed and evaluate an improved protocol, based on the 33 combination of variables from the selected methodologies that demonstrated higher 34 potential for extracting H. commersoni DNA. The quality of samples was assessed through 35 spectrophotometry, gel electrophoresis and PCR-RAPD amplification. The efficiency of 36 DNA extraction was influenced both by the method applied and target-tissue of choice. 37 Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive 38 spectrum of incubation in lysis buffer for up to 36 hours after sample collection, without 39 maceration, using fresh tissues and in the presence of high concentration of Proteinase K. 40 In these conditions, samples were successfully amplified using a set of primers from 41 Operon Technologies. To date, there is no record of description for the parameters 42 analyzed in this work, neither the description of RAPD markers for H. commersoni, 43 evidencing the importance of our findings. 44 45 Key-words: DNA extraction; PCR-RAPD; eye tissue; fresh fish tissue; proteinase K.46 47 48 52 A crucial step for high quality genetic analysis is considered to be a satisfactory DNA 53 extraction. Nevertheless, this purpose is not always easy to achieve, since different 54 biological compounds, such as lipids and proteins, can act as contaminants and interfere in 55 the final quality of the product [1]. The principles and techniques applied in DNA 56 extraction play a pivotal role in obtaining a considerable and purified yield of this molecule 57 [2]. 58A variety of methods has been established to isolate DNA from biological materials 59 [3,6]. Regardless the method of choice, the procedure usually consists of three steps: lysis, 60 purification and DNA recovery, being the first the most critical stage, since is at this point 61 that the chances of causing DNA damages are higher [7]. 62The quality and amount of available DNA strand template have a major influence 63 on genetic analyzes based on PCR (Polymerase Chain Reaction), for example [8,9]. 64 Therefore, an ideal extraction technique should optimize DNA yield, minimize DNA 65 degradation, and be efficient in terms of cost, time, labor, and supplies [10]. 66With the availability of a plethora of molecular analysis machinery, there is an 67 exponential demand for high quality samples, capable to generate great results with 68 minimum of errors. To meet these requirements, commercial kits were developed, aiming 69 for DNA extractions of excellence [11][12][13]. 70