Determination of apolipoprotein E genotypes by single-strand conformational polymorphism

1993 ◽  
Vol 39 (10) ◽  
pp. 2121-2124 ◽  
Author(s):  
M Y Tsai ◽  
P Suess ◽  
K Schwichtenberg ◽  
J H Eckfeldt ◽  
J Yuan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Apolipoprotein E ◽  
Single Strand ◽  
Amplification Refractory Mutation System ◽  
Amino Acid Residues ◽  
Single Strand Conformational Polymorphism ◽  
Conformational Polymorphism ◽  
Apo E ◽  
Apolipoprotein E Genotypes ◽  
Mutation System

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.

Analysis of apolipoprotein E genotypes by the Amplification Refractory Mutation System

1991 ◽  
Vol 37 (2) ◽  
pp. 241-244 ◽  
Author(s):  
PhIlIp R Wenham ◽  
ClIve R Newton ◽  
WillIam H Price
Keyword(s):  
The Other ◽  
Oligonucleotide Primer ◽  
Amplification Refractory Mutation System ◽  
Oligonucleotide Probes ◽  
Post Translational Modification ◽  
Human Dna ◽  
Apo E ◽  
Apolipoprotein E Genotypes ◽  
Mutation System ◽  
Allele Specific

Abstract The Amplification Refractory Mutation System (ARMS) has been successfully applied to the detection of apolipoprotein (apo) E genotypes in human DNA extracted from peripheral blood. By using four allele-specific oligonucleotide primers and one common primer, one can identify the three common alleles of the apo E genetic polymorphism, epsilon 2, epsilon 3, and epsilon 4. The system amplifies two sequences of the apo E gene, one of 181 bp and the other 319 bp. These sequences are amplified when DNA containing a particular allele is incubated with its allele-specific oligonucleotide primer and a common primer. The method is simple, reliable, and nonisotopic and obviates the need for digestion with restriction endonucleases or for hybridization with allele-specific oligonucleotide probes. Genotyping DNA by this method overcomes the problem of post-translational modification of the apo E phenotype encountered with isoelectric focusing of the mature plasma apo E protein.

High-throughput method for determination of apolipoprotein E genotypes with use of restriction digestion analysis by microplate array diagonal gel electrophoresis

1995 ◽  
Vol 41 (11) ◽  
pp. 1599-1604 ◽  
Author(s):  
M K Bolla ◽  
L Haddad ◽  
S E Humphries ◽  
A F Winder ◽  
I N Day
Keyword(s):  
Apolipoprotein E ◽  
High Throughput ◽  
Gel Electrophoresis ◽  
Detection Sensitivity ◽  
High Yield ◽  
Restriction Digestion ◽  
Special Equipment ◽  
Alzheimer Dementia ◽  
Apo E ◽  
Apolipoprotein E Genotypes

Abstract Molecular epidemiological research has identified the association of a common apolipoprotein E (apo E) isoform (E4 as opposed to E3), with risk both of coronary artery disease and of Alzheimer dementia. In addition, the role of apo E genotype (usually E2/E2) in Type III hyperlipidemia is well known. However, both for diagnostic and research purposes, apo E genotyping is cumbersome. The preferred approach is electrophoretic sizing of restriction digestion fragments, enabling simultaneous analysis of the two codons (112 and 158) that represent the six common genotypes (E2/E2; E2/E3; E2/E4; E3/E3; E3/E4; E4/E4). However, the consequent demands of high-yield PCR, high-resolution, high-throughput electrophoresis, and sufficient detection sensitivity have left shortfalls in published protocols. In conjunction with a high-throughput electrophoresis system we described recently, microplate array diagonal gel electrophoresis (MADGE), we have constructed extensively optimized, simplified protocols for DNA isolation from mouthwash samples for PCR setup and high-yield PCR, for restriction digestion, and for subsequent MADGE gel image analysis. The integral system enables one worker to readily undertake apo E genotyping of as many as hundreds of DNA samples per day, without special equipment.

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