FISHERIES RESEARCH INSTITUTE,MOA,TAIWAN

This study was to find an accurate, fast, cheap, stabile and no-toxic genomic DNA extracting method for tilapia. Purity, yield and PCR amplifyied microsatellite and RAPD of extracted DNA from (1) commercial kit method (DNeasy Blood & Tissue Kit, Qiagen); (2) phenol-chloroform method; (3) ammonium acetate method; (4) protein salting-out method and (5) simple boiling method were compared. Methods (2) to (4) were modified to fit the sample.In DNA purity, the phenol-chloroform method and ammonium acetate method were as good as commercial kit method (p>0.05); and the protein salting-out method was worse than phenol-chloroform method and ammonium acetate method (p<0.05), but far higher than simple boiling method (p<0.05). In DNA yield, the simple boiling method was the highest (p<0.05), but its purity was poor. The DNA yield of protein salting-out method and ammonium acetate method were not difference (p>0.05), but higher than commercial kit method and phenol-chloroform method (p<0.05); the ammonium acetate method and phenol-chloroform method were not difference (p>0.05), but higher than commercial kit method (p<0.05).The PCR amplifications of microsatellite UNH106 and GM139 were successful in 30 of 30 (100%) by commercial kit method, phenol-chloroform method, ammonium acetate method, and protein salting-out method, 26 of 30 (86.7%) and 19 of 30 (63.3%) by simple boiling method. The PCR amplifications of RAPD were successful in 30 of 30 (100%) by commercial kit method, phenol-chloroform method, ammonium acetate method, and protein salting-out method, and 21 of 30 (70.0%) by simple boiling method. Although there were 21 samples from simple boiling method that could be amplified from RAPD－PCR, but the number of bands were not enough to run RAPD test.The results showed that protein salting-out method and simple boiling methods were not effectively to extract pure DNA. Among the phenol-chloroform method, ammonium acetate method and commercial kit method, the study would recommend ammonium acetate method due to effective, cheap and no toxic solvent use.