Optimization of DNA Extraction Protocol Using Non-Patented Bulk Reagents

Abstract

DNA-based detection methods for detection of penaeid shrimp viruses are used routinely at present in a number of laboratories around the world. Before going on track in this case extraction of DNA is obvious. Different methods have various effects on DNA extraction. An ideal extraction technique should optimize DNA yield, minimize DNA degradation, and be efficient in terms of cost, time and labor. Therefore which method is optimized one for WSSV infected shrimp among commonly used protocols was indispensable to justify. In the present study shrimp pleopod tissue was used, six protocols were applied and two protocols were optimized for DNA extraction. Three types of DNA extraction kits (Purelink Genomic DNA kit, IQ2000 WSSV Kit, DNAzol) and three types of DNA extraction buffer (0.5% SDS + Proteinase K, 0.5% SDS and 1% SDS) were used. All reagents' cost was analyzed for 200 times extraction. The time was analyzed to finish one extraction (excluding the time of PCR and gel electrophoresis). DNA concentration was higher when Purelink® Genomic DNA kit was used but it was not time saving and procedure was complex than other methods and was an expensive method. Purity of DNA obtained from protocol-6 (DEB-3) was the best and result shows that there was no chance of protein and phenol contamination but it was most expensive method. In the term of cost and time protocol-4 and protocol-5 were standard methods.