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題 名 | 引子選擇及反應緩衝液組成對芥藍幼苗之差異顯示反轉錄-聚合酶連鎖反應譜帶式樣的影響=Effect of Primer Choice and Reaction Buffer Component on the Banding Patterns of Differentially Display RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) in Chinese Kale Seedlings |
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作 者 | 白佳惠; 藍清隆; | 書刊名 | Fu Jen Studies. Science and Engineering |
卷 期 | 31 1997.12[民86.12] |
頁 次 | 頁53-65 |
分類號 | 368 |
關鍵詞 | 芥藍; 差異顯示反轉錄-聚合酶連鎖反應; DNA; RNA; |
語 文 | 中文(Chinese) |
中文摘要 | 應用差異顯示反轉錄-聚合□連鎖反應(differential displayreverse transcription-polymerase chain reaction;DDRT.PCR)系統,以芥藍幼苗時期的總RNA,利用anchored oligo-dT prime:煉合於mRNA的polyadenylotod tail進行反轉錄再和另一條具十個鹼基的逢機引子組合進行聚合□連鎖反應後,於6%DNA定序膠體上依不同片段大小呈現出不同的mRNA衍生物。研究中發現總RNA需先以DNase I處理,而20μM dNTP、1.5mM MgCl□濃度是進行PCR最適當濃度;並可採用DNA銀染(silvor staining)技術快速呈現譜帶式樣。T□MT和不同5'-逢機引子進行DDRT-PCR均呈現高分子量、無法區分的式樣,T□MA、T□MG、T□MC和不同5'-逢機引子進行DDRT-PCR則能呈現50-80個譜帶。 |
英文摘要 | A DDRT-PCR (differential display reverse transcription-polymerase chain reaction) protocol, starting with total RNA from young Chinese kale seedlings, used an anchored oligo-dt primer to anneal to MRNA at the poly-(A) tail for reverse transcription, and a subsequent PCR with the same anchored oligo-dt primer and a random 10-mer primer, was developed to differentially display Chinese kale MRNA species on a 6 % DNA sequencing gel by size. DNA silver staining was used to quickly display the DDRT-PCR products. For the PCR, 20 μM DNTP and 1.5mM MgCl, were optimal. In addition, pre-treatment of total RNA with DNase 1 was necessary. A gel profile of 50-80 bands was normally displayed when, together with a random 10-mer 5'-primer, with one of T□MA, T□MG, and T□MC 3'-primers was used, but not with the T□MT series. |
本系統中英文摘要資訊取自各篇刊載內容。