Genotyping of apolipoprotein A-IV by digestion of amplified DNA with restriction endonuclease Fnu4HI: use of a tailored primer to abolish additional recognition sites during the gene amplification.
Apolipoprotein A-IV (apoA-IV) is involved in the metabolism of chylomicrons and high density lipoproteins. It displays genetic polymorphism due to two co-dominant alleles apoA-IV1 and apoA-IV2. The mutation that causes the polymorphism is a G to T substitution in the third base of codon 360 in the apoA-IV2 allele which results in a glutamine (Gln) to histidine (His) change of amino acid 360. This substitution leads to the abolition of a recognition site for the restriction enzyme Fnu4HI. Part of the third exon of the apoA-IV gene is amplified by the polymerase chain reaction (PCR) using a tailored primer, which abolishes the downstream recognition sites during the DNA amplification. The PCR products are digested with the restriction enzyme Fnu4HI and electrophoresed on a polyacrylamide gel. The apoA-IV genotypes are determined after staining with either ethidium bromide or silver. To validate the method, we determined the inheritance of the apoA-IV alleles in a three-generation kindred of 8 subjects and analyzed amplified DNA of 32 subjects of different apoA-IV phenotypes with this method. The results were compared to those obtained from isoelectric focusing and immunoblotting. In all cases studied, the two methods gave concordant results.
Tenkanen
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National Public Health Institute, Helsinki, Finland.
D001054:Apolipoproteins A / Q000235:genetics*; D001483:Base Sequence; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length
D001054:Apolipoproteins A; C022205:apolipoprotein A-IV; D004247:DNA; C069758:endodeoxyribonuclease Fnu4HI; D015252:Deoxyribonucleases, Type II Site-Specific