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標題: 13C,15N-同位素標定核酸的製備
Preparation of 13C,15N-isotope labeled deoxyribonucleotides
作者: 余龍蟠
Yu, Long-Pan
Contributors: 周三和;楊明德
Ming-Te Yang
國立中興大學
關鍵字: 13C;15N;GST-TMPK;deoxyribonucleotides;phosphorylation;M.methylotrophus;NMR
同位素;激;去氧核醣核酸;磷酸化;核磁共振;甲醇
日期: 2000
Issue Date: 2012-08-31 16:52:54 (UTC+8)
Publisher: 生物化學研究所
摘要: 本實驗參考 Batey 與 Nikonowicz 等人所提出的方法來進行 13C/15N 同位素標定去氧核糖核酸的製備。以 13C/15N 同位素標定核酸所製成的樣品可供多維的異核核磁共振實驗使用。同位素標定可大量減少核磁共振光譜訊息重疊的情形,此對於較大分子量 (大於二十個核酸) 圖譜解讀有很大的幫助。利用 13C-methanol 及 (15NH4)SO4 作為 M. methyltrophus 生長的唯一碳源及氮源,使13C/15N經由ribulose monophosphate pathway而到達核酸的鹼基上。之後經 phenol/chloroform 處理可萃取出 DNA 與 RNA,再經由 RNase 的處理以分解 RNA並取得 DNA,再分別以 DNase I 與 Nuclease P1 作用使其水解成單磷酸狀態。將所要之 dNMPs 收集下來測其濃度後得知每4~5 g的溼菌約可得到約 10~24 mg 的去氧核糖核酸量。最後再進行磷酸化反應,並利用 HPLC 分析其結果。利用此經過標定的 dNTP 進行 PCR或 in vitro transcription,可得到特定序列的 DNA 產物。
在製備 13C/15N 同位素標定的核酸時,中間需有磷酸化的過程。此過程需多種不同的激使 dNMP 變成 dNTP。大部分的激可由市售購得,但從 dTMP 轉變為 dTTP 的專一性磷酸化激必須自行製備。本實驗室利用由陽明大學蘇金源教授所提供含有 cdc8 基因的 pGEX-2T 質體,成功的將之轉型 (transform) 送入E. coli BL21(DE3) 宿主中,並讓此重組蛋白 (GST-TMPK) 於菌體中大量表現後純化,再以 HPLC 分析其酵素活性。
In order to obtain 13C- and 15N-labeled deoxyribonucleotides, we followed and modified the procedure described by Smith et al. in 1997. Preparation of 13C- and 15N-labeled deoxyribonucleotides is necessary to resolve the highly overlapped proton spectrum. First, we obtained pGEX-2cdc8 plasmid from Dr. Su at Yang Ming University. This plasmid contains a cdc8 gene of the yeast Saccharomyces cerevisiae, which encodes the thymidylate kinase (TMPK). This enzyme can specifically phosphorylates dTMP to dTDP, and allow us to prepare in large scale of isotope-labeled dTTP. After transforming this plasmid and over expressing this protein in the host cell (E.coli BL21), we used glutahione-Sepharose column to purify this enzyme and then assayed its activity by HPLC method.
With 13C-methanol and 15N-ammonium sulfate as the sole carbon and nitrogen sources in the culture medium of M. methyltrophus, the bases and riboses of the DNA and RNA could be labeled with 13C/15N. The 13C- and 15N-labeled DNA and RNA were then isolated from the cell culture after being treated with RNase and isopropanol precipitation (instead of Affigel-601 column). More purified DNA could be obtained in this way. We then use DNase and Nuclease P1 to completely hydrolyze the DNA to 5'-monophosphate deoxyribonucleotides. Finally, 5'-monophosphate deoxyribonucleotides were enzymatically phsphorylated to 5'-triphosphate deoxyribonucleotides, which were then used to synthesize a defined DNA sequence by PCR.
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