Characterization of a new human apolipoprotein A-I Yame by direct sequencing of polymerase chain reaction-amplified DNA.
A new genetic variant apolipoprotein (apo), A-I Yame, was discovered during screening for apoA-I genetic variants with isoelectric focusing gel electrophoresis. To investigate the structural abnormality of apoA-I Yame, we amplified the apoA-I gene isolated from the proband's peripheral blood leukocytes with the polymerase chain reaction (PCR) method and directly sequenced these PCR fragments. ApoA-I Yame was found to have aspartic acid (GAC) substituted by tyrosine (TAC) at residue 13. We also identified this substitution by an automated DNA sequencer. This substitution was confirmed with amino acid sequencing of the isolated apoA-I Yame by Immobiline gel electrophoresis. This combined method, direct PCR from genomic DNA-derived individual peripheral blood leukocytes and subsequent direct sequencing, can be used to identify the entire sequence of apoA-I in a short period of time. Furthermore, with this method, it is possible to identify both alleles in heterozygous individuals.
Takada
Y
Y
Department of Internal Medicine, University of Fukuoka School of Medicine, Japan.
Sasaki
J
J
Seki
M
M
Ogata
S
S
Teranishi
Y
Y
Arakawa
K
K
D000483:Alleles; D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D005260:Female; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D007525:Isoelectric Focusing; D008875:Middle Aged; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010375:Pedigree; D016133:Polymerase Chain Reaction
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A