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.
National Public Health Institute, Helsinki, Finland.
H
H
Tenkanen
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