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J Immunol Methods. 2003 Jun 1;277(1-2):185-92.

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

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EA 3249, Group, Laboratoire d'Immunologie, Faculté de Médecine, 2 bis Boulevard Tonnellé, 37032 Cedex, Tours, France.


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.

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