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

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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