Detection for Mutant of Apolipoprotein E Gene by GC-clamp Denaturing Gradient Gel Electrophoresis

载脂蛋白E受体结合区的几种DNA突变常与III型高脂蛋白血症有关。GC-clamp变性梯度凝胶电泳（DGGE）已被用于检测DNA序列的变化。本方法可用于载脂蛋白E异构体的测定或新的载脂蛋白E突变的检测。从200 μ l的全血中提取基因组DNA进行聚合酶链反应（PCR），扩增出一个244 bp的片段，包含91 ~ 165个氨基酸残基。该DNA片段包括受体结合结构域。它还包括112和158个氨基酸残基，其中突变分别表征两种常见变体，apo E2和apo E4。第二个PCR产物用5 ′-引物或3 ′-引物扩增，并附加40-bp的富含G+ C的序列（GC-钳），产物在变性梯度凝胶上在60 ℃水浴中固定。 ...更多信息 5 ′-GC-clamp-DGGE可将E4或E3与E2区分开来。3 ′-GC-clamp-DGGE将E3和E4区分开来。5 ′-和3 ′-GC-clamp-DGGEs联用可测定载脂蛋白E的所有6种表型。此外，还可以检测到载脂蛋白E的罕见突变体--在此基础上，建立了非放射性的单链构象多态性（SSCP）分析方法。第一次PCR产物用0.5M NaOH变性为单链，42 ℃加热，100%甲酰胺溶解，样品在6%聚丙烯酰胺凝胶上4 ℃固定80分钟。然后，用溴化乙锭染色。应用此方法对50例高脂血症患者进行了研究，发现了一个apo E家族-Kochi，并为进一步发现apo E的其他突变提供了可能。少

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