Determination of apolipoprotein E genotypes by single-strand conformational polymorphism

Abstract

We used single-strand conformational polymorphism (SSCP) to determine apolipoprotein E (Apo E) genotypes in 47 individuals. A 295-base-pair (bp) DNA fragment coding for amino acid residues 80-178 of the Apo E protein gave distinct patterns for the three alleles. When we used SSCP to determine the Apo E polymorphism of five individuals whose phenotyping results differed from those of genotyping, the SSCP results agreed with the genotyping results obtained by the PCR-based amplification refractory mutation system (ARMS). Because most of the reported rare alleles of the Apo E gene involve mutations of amino acid residues in positions 120-160, our SSCP method is useful for determining rare as well as common alleles.

Genetic heterogeneity of apolipoprotein E and its influence on plasma lipid and lipoprotein levels

“…Recently, molecular biological techniques such as denaturing gradient gel electrophoresis (DGGE) (194) or single-strand conformation polymorphism (SSCP) analysis (195) followed by direct sequencing have been developed to identify molecular de-fects in the APOE gene (108,196,197…”

<em>Apolipoprotein &epsilon;7 </em>allele in memory complaints: insights through protein structure prediction

“…Several methods have been developed to determine APOE genotypes, such as polymerase chain reaction (PCR)-restriction fragment length polymorphism, 8 – 10 PCR-single strand conformation polymorphism, 11 , 12 and real time PCR. 13 Considering that these methods can misidentify APOE ε7 alleles as ε3 , an evaluation was performed via sequencing analysis to avoid such errors.…”

Rapid determination of apolipoprotein E genotype using a heteroduplex generator

“…For example, although ASO ( 23) is a sensitive method, it is time consuming and requires the labeling of the oligonucleotide probe with radioisotopes (47). SSCP involves the use of radioisotopes or cumbersome silver staining for the detection of band patterns (22), which are also very sensitive to the conditions under which SSCP has been carried out (48). The ARMS method (21) involves no labeling with radioactivity, and gels can easily be stained with ethidium bromide, but for each sample four PCR reactions have to be performed.…”

“…Numerous different methods have already been established for determining the three apoE isoforms. Originally this was carried out at the protein level by isoelectric focusing (IEF) (20), and more recently at the DNA level by methods such as amplification refractory mutation system (ARMS) (21), single strand conformation polymorphism (SSCP) (22), allele-specific oligonucleotide probes (ASO) (23), and restriction enzyme analysis (24). Numerous improvements to these methods have been described (25)(26)(27)(28)(29)(30), but all of these have disadvantages.…”