Abstract
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.
Highlights
DNA was isolated from 5 ml of EDTA blood from subjects belonging to different Apolipoprotein A-IV (apoA-IV) phenotypes [20]
Genomic DNA (0.2 pg) isolated from apoA-IV 1-1, 1-2, and 2-2 subjects was amplified by the polymerase chain reaction [21]
The PCR was performed in a reaction mixture containing 0.5 pM of primers, 0.2 mM each of dATP, dCTP, dGTP, and Abbreviations: apoA-IV, apolipoprotein A-IV, Gln, glutamine; His, histidine; HDL, high density lipoproteins; IEF, isoelectric focusing; PCR, polymerase chain reaction; bp, base pairs
Summary
Phenotype analysis was performed by immunoblotting after isoelectric focusing of serum [15]. DNA was isolated from 5 ml of EDTA blood from subjects belonging to different apoA-IV phenotypes [20]. Genomic DNA (0.2 pg) isolated from apoA-IV 1-1, 1-2, and 2-2 subjects was amplified by the polymerase chain reaction [21]. The primers were chosen according to the published apoA-IV gene structure [22], I: 5'-CCT GAG GGA CAA G G T CAA C E - 3 ' (nucleotides 2285-2305), 11: 5'-CAC CTG CTC CTG CTA* CTG C E C-3'. The PCR was performed in a reaction mixture containing 0.5 pM of primers, 0.2 mM each of dATP, dCTP, dGTP, and Abbreviations: apoA-IV, apolipoprotein A-IV, Gln, glutamine; His, histidine; HDL, high density lipoproteins; IEF, isoelectric focusing; PCR, polymerase chain reaction; bp, base pairs
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