High-throughput method for determination of apolipoprotein E genotypes with use of restriction digestion analysis by microplate array diagonal gel electrophoresis

Clin Chem. 1995 Nov;41(11):1599-604.

Abstract

Molecular epidemiological research has identified the association of a common apolipoprotein E (apo E) isoform (E4 as opposed to E3), with risk both of coronary artery disease and of Alzheimer dementia. In addition, the role of apo E genotype (usually E2/E2) in Type III hyperlipidemia is well known. However, both for diagnostic and research purposes, apo E genotyping is cumbersome. The preferred approach is electrophoretic sizing of restriction digestion fragments, enabling simultaneous analysis of the two codons (112 and 158) that represent the six common genotypes (E2/E2; E2/E3; E2/E4; E3/E3; E3/E4; E4/E4). However, the consequent demands of high-yield PCR, high-resolution, high-throughput electrophoresis, and sufficient detection sensitivity have left shortfalls in published protocols. In conjunction with a high-throughput electrophoresis system we described recently, microplate array diagonal gel electrophoresis (MADGE), we have constructed extensively optimized, simplified protocols for DNA isolation from mouthwash samples for PCR setup and high-yield PCR, for restriction digestion, and for subsequent MADGE gel image analysis. The integral system enables one worker to readily undertake apo E genotyping of as many as hundreds of DNA samples per day, without special equipment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apolipoproteins E / genetics*
  • Base Sequence
  • DNA / chemistry
  • DNA / isolation & purification
  • Deoxyribonucleases, Type II Site-Specific*
  • Electrophoresis / methods*
  • Genotype*
  • Humans
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Saliva / chemistry
  • Templates, Genetic

Substances

  • Apolipoproteins E
  • DNA
  • Deoxyribonucleases, Type II Site-Specific
  • GCGC-specific type II deoxyribonucleases