Detection for Mutant of Apolipoprotein E Gene by GC-clamp Denaturing Gradient Gel Electrophoresis
GC夹变性梯度凝胶电泳检测载脂蛋白E基因突变
基本信息
- 批准号:03671155
- 负责人:
- 金额:$ 1.09万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for General Scientific Research (C)
- 财政年份:1991
- 资助国家:日本
- 起止时间:1991 至 1992
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
It has been reported that several DNA mutations in the receptor binding domain of apolipoprotein E(apo E)are usually involved in type III hyperlipoproteinemia. GC-clamp denaturing gradient gel electrophoresis(DGGE)has been applied for the detection of DNA sequence changes. We established this method for determination of apo E isoforms or detection of new apo E mutation. Genomic DNA for polymerase chain reaction(PCR)was extracted from whole blood of 200 ul.A244-bp fragment containing amino acid residues from 91 to 165 was amplified by PCR. This DNA fragment included the receptor binding domain. It also included 112 and 158 amino acid residues of which mutations characterize the two common variants,apo E2 and apo E4,respectively. The second PCR product was amplified with 5'-primer or 3'-primer which had been attached with a 40-bp G+C-rich sequence (GC-clamp),and the product was electrophoresed on a denaturing gradient gel in a bath at 60゚C temperature.The gel consisted of 7% polyacrylami … More de with a linearly increasing gradient from 60%to80% denaturant(100% denaturant:7 M urea/40% formamide)for 5'-GC-clamp or from 50%to70% denaturant for 3'-GC-clamp.After electrophoresis,the gel was stained with ethidium bromide.The5'-GC-clamp-DGGE made possible to distingish E4 or E3 from E2. The3'-GC-clamp-DGGE distinguished E3 from E4. All six phenotypes of apo E could be determined by the combination of 5'-and3'-GC-clamp-DGGEs. Additionally,apo E-Kochi(145Arg->His),rare mutant of apo E,could be detected by using these methods. Further more,we established a single-strand conformation polymorphism(SSCP)which is non-radioactive method. The first PCR product was denatured to single-strand by 0.5 M NaOH and heating at 42゚C,and was resolved with 100% formamide.The sample was electrophoresed on 6% polyacrylamide gel at 4゚C for 80 minutes. Then,the gel was stained by ethidium bromide. The six phenotypes of apo E were clearly distinguished by this method.SSCP method for apo E analysis was simpler and more sensitive than DGGE.And PCR-SSCP needed only6hours for the analysis.Fifty patients with hyperlipoproteinemia were studied by these two methods,and another family of apo E-Kochi was found.It is possible to find other mutations of apo E by these methods. Less
据报道,载脂蛋白E(apo E)受体结合域的几种DNA突变通常与III型高脂蛋白血症有关。GC-clamp变性梯度凝胶电泳(DGGE)已被应用于DNA序列变化的检测。我们建立了载脂蛋白E同型体的测定或新的载脂蛋白E突变的检测方法。从200 ul全血中提取基因组DNA用于聚合酶链反应(PCR)。通过PCR扩增出含有91 ~ 165个氨基酸残基的A244-bp片段。该DNA片段包含受体结合域。它还包括112和158个氨基酸残基,这些氨基酸残基的突变分别表征了两种常见变异,载脂蛋白E2和载脂蛋白E4。用5′引物或3′引物扩增,引物上附40 ~ bp的G+C-rich序列(GC-clamp),并在60℃的温度下在变性梯度凝胶上电泳。5'-GC-clamp的凝胶由60%到80%的变性剂(100%变性剂:7 M尿素/40%甲酰胺)线性增加,3'-GC-clamp的变性剂从50%到70%线性增加。电泳后用溴化乙锭染色。5′-GC-clamp-DGGE可以区分E4或E3与E2。3′-GC-clamp-DGGE区分了E3和E4。载脂蛋白E的所有六种表型都可以通过5'-和3'-GC-clamp-DGGEs的组合来确定。此外,这些方法还可以检测到罕见的载脂蛋白E突变体apo E- kochi (145Arg->His)。此外,我们还建立了一种非放射性的单链构象多态性(SSCP)方法。第一个PCR产物在0.5 M NaOH和42ºC加热下变性为单链,用100%甲酰胺溶解。样品在6%聚丙烯酰胺凝胶上电泳,温度为4 /丙油,电泳时间为80分钟。然后用溴化乙锭染色。通过这种方法可以清楚地区分载脂蛋白E的六种表型。SSCP法测定载脂蛋白E比DGGE法简单、灵敏。而PCR-SSCP分析只需要6小时。用这两种方法对50例高脂蛋白血症患者进行了研究,发现了另一个载脂蛋白E-Kochi家族。通过这些方法可以发现载脂蛋白E的其他突变。少
项目成果
期刊论文数量(5)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
末廣 正: "PCRとGDクランプ法を用いたアポリポ蛋白E遺伝子の変異種の発見" 動脈硬化. 19. 1015 (1991)
Tadashi Suehiro:“使用 PCR 和 GD 钳法发现载脂蛋白 E 基因变异”动脉硬化。 19. 1015 (1991)
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
安岡 伸和: "PCRとGCクランプ法を用いたアポリポ蛋白E遺伝子の変異種の発見" 動脈硬化. 19. 1015 (1991)
Nobukazu Yasuoka:“使用 PCR 和 GC 钳夹方法发现载脂蛋白 E 基因的变体”动脉硬化。
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
Nobukazu Yasuoka: "Detection for Mutant of Apolipoprotein E Gene by GC-clamp Denaturing Gradient Gel Electrophoresis" J.Jpn.Atherosclerosis Soci. 19. 1015 (1991)
Nobukazu Yasuoka:“通过 GC 钳变性梯度凝胶电泳检测载脂蛋白 E 基因突变”J.Jpn.Atherosclerosis Soci。
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- 发表时间:
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- 影响因子:0
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Nobukazu Yasuoka: "Detection of apolipoprotein E variant by GC‐clamps and denaturing gradient gel electrophoresis" International Atherosclerosis Society 59th E.A.S.Congress Abstructs.93 (1992)
Nobukazu Yasuoka:“通过 GC 夹和变性梯度凝胶电泳检测载脂蛋白 E 变体”国际动脉粥样硬化协会第 59 届 E.A.S.Congress Abstracts.93 (1992)
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SUEHIRO Tadashi其他文献
SUEHIRO Tadashi的其他文献
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{{ truncateString('SUEHIRO Tadashi', 18)}}的其他基金
Role of Paraoxonase in Diabetic Vascular Complications by Oxidative Stress
对氧磷酶在氧化应激引起的糖尿病血管并发症中的作用
- 批准号:
11671125 - 财政年份:1999
- 资助金额:
$ 1.09万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Spin Distribution and Chemical Reactivity of sigma-Free Radicals
西格玛自由基的自旋分布和化学反应性
- 批准号:
02640404 - 财政年份:1990
- 资助金额:
$ 1.09万 - 项目类别:
Grant-in-Aid for General Scientific Research (C)