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研究生:曾瑞泰
研究生(外文):Tzeng, Ruey-Tay
論文名稱:AntisenseDNA的毛細管電泳分離
論文名稱(外文):The Separation of Antisense DNA by Capillary Electrophoresis
指導教授:陳淑慧陳淑慧引用關係
指導教授(外文):Shu-Hui Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
中文關鍵詞:毛細管電泳
外文關鍵詞:Antisense DNA
相關次數:
  • 被引用被引用:0
  • 點閱點閱:204
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    Antisense DNA做為病毒和疾病的治療藥物近年來
已進入臨床實驗階段。本論文利用聚合物溶液作為methyltran
sferase antisenseDNA(MT-AS)的分離介質
應用於毛細管膠電泳上。針對所有影響分離的參數作有系統的研究和討論
。    首先針對poly(deoxyadenylic acid
)pd(A)19,20和未修飾的antisense DNA 15
-20mers在不同的緩衝溶液系統(100mM ammonium
formate, pH4.5和100mM Tris-borate
,pH8.2)、變性試劑(urea)和溫度下的分離作討論。其中發
現pd(A)19,20可以在100mM ammonium for
mate(pH4.5)下成功地分離,然而未修飾的antisens
e DNA15-20mers則無法分離。結果顯示13% (w/w
) poly(ethylene glycol) (PEG)聚合物
溶液看來是可置換的而且適用於antisense DNA 15-2
0mers的分離。使用衍生化毛細管柱在於未修飾的antisens
e DNA的分離上可以減少管壁的吸附作用,而且可以快速地分離未修
飾的antisense DNA 15-20mers。然而對於以硫
修飾的antisense DNA除了使用衍生化毛細管柱外,發現f
ormamide對於達到成功地分離也是同等的重要。實驗結果顯示,
利用13% (w/w) PEG形成動態網篩方式可達到antise
nse DNA 15-20mers單一鹼基間分離的效果。    
將所發展之方法應用於生物檢體基質的定性及定量分析,探討基質對於注
射方式和電泳分離的影響。以pd(A)10當內標準品所建立的標準曲
線對以硫修飾的antisense DNA之線性濃度範圍可由5pp
m至150ppm (R2=0.99),其遷移時間和波峰面積的相對
標準偏差範圍分別為0.02-1.2 %和1.7-26.5 %。以
3倍的訊雜比所得到的偵測極限為1ppm。本實驗顯示PEG聚合物溶
液可提供在與antisense DNA有關之基礎研究和藥物研究上
更便利的方法。
    The use of antisense DNA as 
the therapeutic agent against vi
rus or disease-causing genes has
 been undergoing the clinical tr
ials.  In this study, the polyme
r solution is used as the separa
tion medium in capillary gel ele
ctrophoresis for the separation
 of methyltransferase antisense 
DNA (MT-AS). All separation para
meters are systematically invest
igated and discussed.    First, 
the separation of poly(deoxyaden
ylic acid) pd(A) 19,20 and unmod
ified antisense DNA 15-20mers un
der different buffer systems (10
0mM ammonium formate, pH4.5 and 
100mM Tris-borate, pH8.2), denat
uring agents (urea) and temperat
ures were explored. It was found
 that pd(A) 19,20 can be separat
ed under 100mM ammonium formate 
(pH4.5).  Whereas unmodified ant
isense DNA could not be resolved
. The solution of 13% (w/w) poly
(ethylene glycol) (PEG) appears 
to be replenishable and suitable
 for the separation of 15-20mers
 of antisense DNA. The capillary
 column was further derivatized 
and used for the separation of u
nmodified antisense DNA to reduc
e the column adsorption. The 15-
20mers of unmodified antisense D
NA were readily separated by the
 use of the derivatized column.
 For modified antisense DNA, how
ever, formamide was found to be 
important in addition to the use
 of a derivatized column to achi
eve the separation. The experime
ntal result indicates that the d
ynamic sieving medium constitute
d by 13% (w/w) PEG can be used t
o reach the resolution of 15-20m
ers of antisense DNA.    The dev
eloped method was applied for th
e qualitative and quantitative a
nalysis of biological samples an
d the effect of matrix on inject
ion modes and electrophoretic se
paration were examined. With the
 use of pd(A)10 as the internal 
standard, a linear calibration c
urve was constructed with the co
ncentration of standard solution
s ranging from 5 to 150 ppm (R2 
= 0.99). The relative standard d
eviation of migration time and p
eak area range from 0.02 to 1.2 
% and 1.7 to 26.5 %, respectivel
y. The detec?ion limit was deter
mined to be 1 ppm with a S/N rat
io of 3. Based on this study, th
e PEG solution appears to provid
e a more convenient mean for the
 general research and pharmaceut
ical studies on antisense DNA.

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