Journal of Lipid Research, Vol 32, 545-549, Copyright © 1991 by Lipid Research, Inc.
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
H Tenkanen
National Public Health Institute, Helsinki, Finland.
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.
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