農業部-水產試驗所全球資訊網農業部-水產試驗所全球資訊網

本研究以吳郭魚作為試驗生物材料，研發準確、快速、便宜、穩定且無毒性的基因組DNA萃取方法，以商業套組法 (DNeasy Blood & Tissue Kit, Qiagen)、酚－氯仿法 (phenol-chloroform method)、醋酸銨法 (ammonium acetate method)、蛋白質析出法 (protein salting-out method) 及簡單加熱法 (simple boiling method) 等5種方法萃取DNA，比較所萃取的DNA純度及產量，以及在微隨體 (microsatellite) 和隨機擴增片段多形性DNA (random amplified polymorphism DNA, RAPD) 等PCR擴增分析應用上有無差異。比較5種萃取方法所得之結果，在DNA純度方面，商業套組法、酚－氯仿法及醋酸銨法3者OD比值並沒有顯著差異 (p > 0.05)，但皆顯著高於蛋白質析出法和簡單加熱法 (p < 0.05)；而蛋白質析出法OD比值顯著高於簡單加熱法 (p < 0.05)。至於5種萃取方法在DNA產量上，簡單加熱法DNA產量顯著高於其他4種方法　(p < 0.05)，但其純度差，可能導致產量上的錯估；蛋白質析出法產量和醋酸銨法沒有顯著差異 (p > 0.05)，但顯著高於商業套組法和酚－氯仿法 (p < 0.05)；醋酸銨法產量和酚－氯仿法沒有顯著差異 (p > 0.05)，但顯著高於商業套組法 (p < 0.05)；而酚－氯仿法產量和商業套組法沒有顯著差異 (p > 0.05)。微隨體PCR擴增UNH106及GM139 DNA兩種片段結果顯示，以商業套組法、酚－氯仿法、醋酸銨法及蛋白質析出法所萃取的DNA，經PCR擴增兩種片段，成功率皆100% (30/30)；簡單加熱法之成功率則分別為86.7% (26/30) 及63.3% (19/30)。RAPD-PCR擴增DNA片段結果，以商業套組法、酚－氯仿法、醋酸銨法及蛋白質析出法所萃取的DNA，經PCR擴增條帶之成功率皆為100% (30/30)；簡單加熱法成功率則為70.0% (21/30)；簡單加熱法在30個DNA樣本中，雖然有21個樣本能擴增出條帶，但其所擴增的條帶數並不足以進行RAPD的分析。本研究所建立的操作技術在酚－氯仿法及醋酸銨法能達到商業套組法的水準，而醋酸銨法的使用又能避免使用具毒性的酚及氯仿，在萃取高純度的DNA方面是值得推薦的有效方法。此外，蛋白質析出法和簡單加熱法，具有經濟、快速、安全及簡便性，惟DNA純度無法達到商業套組法的水準。

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.