頁籤選單縮合
題名 | 盤固草逢機增殖核苷酸多型性(RAPD)分析效率的改進 |
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作 者 | 陳嘉昇; 王紓愍; 成游貴; | 書刊名 | 畜產研究 |
卷期 | 28:4 1995.12[民84.12] |
頁次 | 頁235-244 |
分類號 | 434.4 |
關鍵詞 | 指草; 逢機增殖核苷酸多型性; 去氧核糖核酸萃取; 聚合酶連鎖反應條件; Digitgrass; RAPD; DNA extraction; PCR procedures; |
語文 | 中文(Chinese) |
中文摘要 | 本試驗以指草為材料,探討染色體組DNA萃取步驟的簡化及PCR反應時間的縮短,以提高RAPD分析效率,使更有利於植物遺傳、育種及分類研究之用。依快速的DNA萃取法,只需20mg的植物材料,加入Tris-SDS或CTAB緩衝液以均質機研磨萃取,不須經過65℃水浴振盪,即可獲得足夠進行300次RAPD分析之染色體組DNA,但以CTAB緩衝液萃取時不能省略chloroform/isoamylalcohol分溶的步驟,否則將影響DNA產量。適當的調整PCR反應時之變性、結合及DNA聚合?作用的條件,可有效縮短RAPD分析時間,並且不影響環帶訊息與再現性。調整條件因使用之PCR機型而異,適宜於Perkin-Elmer Cetus DNA thermal cycler-480的條件為:94℃ 20sec, 34℃ 1 min, 72℃ 30 sec共進行35個循環;Idaho-1605 air thermal cycler的條件為:92℃ 0 sec, 40℃ 20 sec, 72℃ 90 sec進行45次循環,同時RAPD圖譜不因機型不同而異。 |
英文摘要 | The purpose of this investigation was to simplify DNA extraction and PCR procedures for improving the efficiency of RAPD analysis in digitgrass. DNA extracted from 20 mg leaves by he rapid method which were directly homogenized in Tris-SDS or CTAB buffer without shaking in 65℃ water bath, was enough for 300 times RAPD analysis. However, if CTAB was used as DNA extraction buffer, procedure of chloroform/isoamylalcohol partion would be necessary. The optimal PCR procedures performed in Perkin-Elmer Cetus DNA thermal cycler-480 was 35 cycles of 94℃ 20 sec, 34℃ 60 sec, and 72℃ 30 sec and in Idaho-1605 air thermal cycler was 45 cycles of 0 sec at 92℃, 20 sec at 40℃, and 90 sec at 72℃. The RAPD pattens amplified in two kinds of DNA thermal cyclers were the same. The results showed that the simplified procedures were much more efficient than before. It would be useful for other species in RAPD analysis. |
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