Genotyping of apolipoprotein E by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS). The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I (Clostridium formicoaceticum), for generating restriction fragments for rapid and accurate mass analysis. An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis. By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved. Genotyping of an unknown sample and obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine analysis of apo E.
Srinivasan
J R
JR
Department of Chemistry, University of Michigan, Ann Arbor 48109, USA.
Kachman
M T
MT
Killeen
A A
AA
Akel
N
N
Siemieniak
D
D
Lubman
D M
DM
D001057:Apolipoproteins E / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000032:analysis; D005092:Exonucleases; D005838:Genotype; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001057:Apolipoproteins E; D004247:DNA; D005092:Exonucleases