Genotyping HLA-B locus by RT-PCR with fret probes

• Published in: Genes and immunity Vol. 6; p. S51
• Main Authors: Faner, R, Casamitjana, N, Coll, J, Llop, C, Pujol-Borrell, R, Palou, E, Juan, M
• Format: Journal Article
• Language: English
• Published: 01-04-2005
• ISSN: 1466-4879

The aim of this work is the use of FRET hybridization probes for HLA-B genotyping, achieving a medium-low resolution with a great reduction of tubes, probes and time consuming procedures. Our strategy defines HLA-B alleles by the cross-information of group-specific amplifications and specificities of probe hybridizations. Our approach next to group-specific primers and probes co-amplifies another gene (beta-globin) as internal control. Therefore monitoring of both amplifications is achieved by application of two sets of FRET probes, one specific for the HLA-B locus (LC-Red 640) and the second set (LC-Red705) for the detection of the internal control gene. In each tube HLA-B probes show different mismatches with the amplified alleles depending on their sequence, giving 2-5 differences for each tube. By the analysis of the melting curve pattern of all the HLA-B probes, a specific genotyping for each sample can be raised with a medium-low final resolution. After successfully set up the approach by testing 250 samples, we plan to type additionally two hundred clinical samples that were previously typed by standard PCR-based methods. In all this pre-typed samples, results were concordant. Moreover, even a reduction in ambiguous results was obtained when using this new approach. In fact, our proposal for defining HLA-B alleles by real-time PCR with FRET probes was found faster and less cumbersome than PCR-SSP or PCR-SSO techniques currently used in HLA genotyping.