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cDNA 編目旨在於瞭解基因表現和轉錄基因組的組成, 是研究分子生物學
寶貴的基礎資源. 本研究應用 Liang 和 Pardee 發展出的差異顯示反轉
錄-聚合酵素連鎖反應(DDRT-PCR, diffrential display reverse
transcription -polymerase chain reaction)系統建立芥藍幼苗時期的
cDNA 庫. DDRT-PCR 主要以總 RNA 利用 anchored oligo-dT primer 煉
合於 mRNA 的 polyadenylated tail 進行反轉錄, 再和另一具10 個鹼基
的逢機引子組合進行聚合酵素連鎖反應, 於6% DNA定序膠體上依不同片段
大小呈現出不同的 mRNA. 本研究中發現總 RNA 需先以 DNase I 處理,
20 uM dNTP, 1.5 mM 鎂離子濃度是聚合酵素連鎖反應的最適當濃度, 並
可採用 DNA 銀染技術快速呈現譜帶式樣. T12MT 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, T12MA, T12MG, T12MC
和不同 5' 逢機引子進行 DDRT-PCR 則能呈現 50-80 個譜帶.於 DNA 定
序上, 同時利用 Pharmacia 自動化定序系統和 GATC 直接轉印電泳/DNA
定序系統針對 29 個芥藍 cDNA 殖株定序, 並以網際網路經 NCBI(
National Center for Biotechnology Information) 伺服器利用
BLASTN 程式分析, 比對定序的 29 個 cDNA 殖株. 經過初步編目, 判定
66% cDNA 殖株的來源確實為 mRNA, 但 34% 無法判斷. 編目結果確定有
一個 cDNA 殖株來源為甘藍類(Brassica oleracea) mRNA, 5 個 cDNA殖
株來源和阿拉伯芥 (Aravidopsis thaliana) 者相似, 3 個 cDNA 殖株無
任何相似性比對結果.根據本研究, 殘留的 DNA, rRNA 等會干擾 DDRT-
PCR反應,致使同一 mRNA 來源的 cDNA 無法以同一種片段大小呈現, 造
成 cDNA 編目上的困擾, 也會於偵測不同型態細胞基因表現差異時表現偽
陽性, 因此欲建立一良好的 DDRT-PCR 系統, 總RNA 的品質, rRNA 的去
除是研究成敗之關鍵;但如何去除 rRNA, 同時不致損失稀有 mRNA 則仍待
克服. 此外, 選用 one-base anchored oligo-dT primer, 降低聚合酵素
連鎖反應煉合溫度,應用非變性膠體也是未來研究改進的方向.
Cataloguing of cDNAs, with the view of better understanding of
gene expression and organization of transcribed genome, is an
invaluable resource for molecular biology research. In this
study, the DDRT-PCR (differential display reverse
transcription- polymorase chain reaction ) developed by Liang
and Pardee was to construct a cDNA library from young kailan
seedling. DNA silver staining was used to quickly display the
DDRT-PCR products. For the PCR, 20 uM dNTP and 1.5 mM MgCl2
were optimal. In addition, pre-treatment of total RNA with
DNase I was necessary. A gel profile of 50-80 bands was
normally displayed when either of T12MA, T12MG,T12MC 3' primer
was used, together with a random 10-mer 5' primer, but not that
with T12MT primer. Sequences of 29 cDNA clones were analyzed
and compared using BLASTN algorithm, to known genome database
at the NCBI server. Totally, 66% of the cDNA clone shows strong
similarity with one or more mRNAs in database, with one cDNA
clone is homologous to Brassica oleracea mRNA and five clones
are homologous to five different Arabidopsis thaliana cDNA
clones, and three cDNA clones are homologous to none of the
known clones. From our DNA sequencing anaoysis, DNA, rRNA could
potentially be the random primer to display different cDNA
fragments resulted from the same mRNA species in the DDRT- PCR,
which in term increasing the amount of DNA sequencing effort
and resulting in false positive results in displaying gene
expression of different cell types. Therefore, preparetion of
total RNA with high quality and elimination of rRNA are the key
issues of establishing a good DDRT-PCR system. However,
protocol to remove rRNA and to retain rare mRNA simultaneously
remains to be developed.Furthermore, alrenative such as the use
of one-base anchored oligo-dT primers and nondenatuered gels,
decrease in annealing temprature in the PCR need to be explored
in the future study.
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