Rapid single-step FCGR3A genotyping based on SYBR Green I fluorescence in real-time multiplex allele-specific PCR.
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.
Dall'Ozzo
Sébastien
S
EA 3249, Group, Laboratoire d'Immunologie, Faculté de Médecine, 2 bis Boulevard Tonnellé, 37032 Cedex, Tours, France.
Andres
Christian
C
Bardos
Pierre
P
Watier
Hervé
H
Thibault
Gilles
G
D000483:Alleles; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines; D017452:Receptors, IgG / Q000737:chemistry / Q000235:genetics*; D015203:Reproducibility of Results
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D017452:Receptors, IgG; C098022:SYBR Green I