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第四节 载脂蛋白CⅢ基因型检测
http://www.100md.com 《动脉粥样硬化》

载脂蛋白CⅢ基因位于11号染色体与载脂蛋白AⅠ-AⅣ形成一基因族,它位于载脂蛋白AⅠ下游,有三个内含子和四个外显子。目前对载脂蛋白CⅢ基因3'末端非编码区C-G对换产生一个Sac-Ⅰ酶切位点与冠心病的关系未有定论。用多聚酶链反应(PCR)扩增载脂蛋白CⅢ基因3'末端233bp,再用Sac-1酶切扩增产物,可以检测载脂蛋白CⅢ基因3'末端的突变及其与动脉粥样硬化相关性。本节采用PCR-RFLP检测ApoCⅢ基因型。

一、仪器与试剂

1.仪器:电泳仪,基因扩增仪。

2.试剂:TaqDNA聚合酶,dNTP,DNA参考物,Sca-1酶。

二、操作方法

1.模DNA提取

取用EDTA抗凝全血100μl加试剂(0.32mol/L蔗糖,5mmol/l MgCl2,1% TritonX-100,0.01mol/L Tris-HClpH7.6),振摇至溶解均匀透明,3000r/min离心10min,弃上清,沉淀部分加0.9NaCl溶液洗一次,加预冷试剂Ⅱ(75mmol/l NaCl,24mmol/L EDTA)100μl,20%SDS15μl,蛋白酶K10μl(6g/L)于65℃水浴4h,加200μl酚和氯仿:异戊醇(24:1)各提两次,上清加无水乙醇1.0ml放置于-20℃2h,以12kr/min离心5min,真空干燥后加50μlTE缓冲液溶解,4℃保存备用。

2.多聚酶链反应

Primer1:5'-GTG ACC GAT GGC TTC AGT TCC CTG-3',primer2:5'-GGt AGG AGA GCA CTC AGA ATA CA-3'。反应总体积为50μl,扩增缓冲液所含物质终浓度为:Tris-HCl,pH8.3,67mmol/L;MgCl225mmol/L;KCl 50mmol/L;明胶0.01%,dNTP各0.2~0.5μmol/L,模板约10μg,加40μl液体石蜡,94℃预变性4min,加Tag聚合酶1.5U,以93℃1min、65℃1.5min、72℃1.5min,循环30次。

3.扩增产物酶切

取扩增产物20μl加入NH4AC20μl,再加300μl无水乙醇混均置-20℃1h,10000r/min离心7min弃上清,干燥后加Sac-1酶液10μl37℃保温2h。

4.扩增产物碱基对分子量测定

采用GB公司产DNa marker,以已知DNA片段bp数相对应的电泳迁移距离,取半对数作图制成标准曲线,再根据等测定扩增产物DNA片段移动距离在标准曲线上求出其bp数。

三、结果分析

采用本节设计引物扩增产物为ApoCⅢ基因3'非编码区233bp,该产物经Sac-1酶切可出现三种结果;①酶切后仍为一条带,电泳迁移率未发生改变,为S1S1纯合子;②酶切后除原始带外又增加了两条带,查标准DNa marker曲线在158bp和75bp相同,为S2S2杂合子;③酶切后原始带消失,出现两条新带,其电泳迁移率与158bp和75bp相同,为S2S2纯合子。S2为扩增的基因产物中C-G对换增加一个酶切位点。

检查100名正常人中有S2杂合子28名,纯合子2名;68名冠心病人中S2杂合16名,纯合子4名,它们的频率变化见表(19-4)。S2基因频率两组间无明显差别。

表19-4 载脂蛋白CⅢ基因Sac-Ⅰ酶切频率表

级别n基因型频 率
S1S1S1S2S2S2S1S2
正常人100702820.840.16
冠心病68481640.820.176

X=0.079,p>0.05(两组比较)

(徐琼)

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