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頁籤選單縮合
題 名 | 吳郭魚基因組DNA萃取方法的比較=Comparison of Methods for Genomic DNA Extraction from Tilapia |
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作 者 | 余俊欣; 曾福生; 朱惠真; 周賢鏘; 盧民益; 林金榮; | 書刊名 | 水產研究 |
卷 期 | 18:1 2010.06[民99.06] |
頁 次 | 頁33-45 |
分類號 | 439.5 |
關鍵詞 | 吳郭魚; DNA萃取; 微隨體; 隨機擴增片段多型性DNA; Tilapia; DNA extraction; Microsatellite; RAPD; |
語 文 | 中文(Chinese) |
中文摘要 | 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。 |
英文摘要 | 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. |
本系統中英文摘要資訊取自各篇刊載內容。