Isolation of Agarose and its Application as medium of Gel ElectrophoresisMethod for HPV (Human papillomavirus) DNA Identification

摘要

Agarose had been separated from commercial agar that was extracted from red algae, Gracilaria gigas. Agarose was obtained by dissolving the agar powder with 70 °C preheated ethylene glycol under continuous stirring followed by increasing the temperature to 105 °C to get homogenous solution with concentration of 1, 2, and 3 % b/v agar. Agarose was separated from more soluble agaropectin by cooling process overnight at -10 °C, followed by adding isopropanol at room temperature to induce precipitation of agarose. Agarose precipitate was then filtered with flannel and dried at room temperature until the smell of isopropanol disappeared and the agarose granule was formed. The granule then was grinded to yield purified agarose powder. Purified agaroses were named agarose A, agarose B, and agarose C. Some chemical and physical properties of isolated agarose such as gelling and melting point, gel strength, sulfate content, and EEO were measured and determined utilizing standard measurement method. It was found that the sulfate content of agarose A, B, and C were 0.13%, 0.16, and 0.22 % respectively; electro-endosmosis (EEO) values were 0.18, 0.29, and 0.36 respectively; and gel strength (1.5 %) were 1594, 1406, and 1219 g/cm² respectively. In this study, isolated agarose gel and agarosa gel standard Thermo Scientific (TopVision®) were used for separation and identification of HPV DNA fractions by gel electrophoresis method. The result showed that banding pattern of isolated agarose gel was close to banding pattern of TopVision® agarose gel. It can be concluded that agarose can be separated from agar using ethylene glycol as a solvent and isopropanol to induce precipitation of agarose.