Rapid single-step FCGR3A genotyping based on SYBR Green I fluorescence in real-time multiplex allele-specific PCR

J Immunol Methods. 2003 Jun 1;277(1-2):185-92. doi: 10.1016/s0022-1759(03)00123-6.

Abstract

A polymorphism at position 559 in the cDNA of the FCGR3A gene encoding the FcgammaRIIIa generates two allotypes with either a valine (Val) or a phenylalanine (Phe) at amino acid position 158. This polymorphism is of major importance in immunopathology and in pharmacogenetics, especially monoclonal antibody treatments. In this study, we report a single-step and single-tube method for FCGR3A-158V/F genotyping by real-time multiplex allele-specific PCR and melting curve analysis in the presence of SYBR Green I fluorescent dye. Results obtained from 113 samples showed 100% accuracy compared to those obtained with a conventional PCR-based allele-specific restriction assay. Although this method requires expensive equipment, it is inexpensive in terms of consumables. It is also very rapid, reliable and suitable for large-scale analysis.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles*
  • Fluorescent Dyes / metabolism*
  • Genotype
  • Humans
  • Organic Chemicals*
  • Polymerase Chain Reaction / methods*
  • Polymorphism, Single Nucleotide
  • Receptors, IgG / chemistry
  • Receptors, IgG / genetics*
  • Reproducibility of Results

Substances

  • Fluorescent Dyes
  • Organic Chemicals
  • Receptors, IgG
  • SYBR Green I