Comparison of two HLA‐DRB high resolution microtiter plate reverse hybridization typing methods: advantage of a codon‐86 valine or glycine PCR segregation

Abstract

Two rapid, nonisotopic, high-resolution HLA-DRB typing methods have been developed for DRB1, DRB3, DRB4 and DRB5 alleles. These methods are based on a single procedure consisting of the reverse hybridization of biotinylated amplicons to oligonucleotide probes that are covalently attached to a microtiter plate. Detection is by an enzymatic reaction with a fluorescent substrate. The 1 Generic Amplification (1GA) method amplifies all HLA-DRB alleles in the same reaction mix. The 2 Allelic Subset Amplification (2SA) method uses two distinct amplification reactions that distributes all DRB alleles into two equal-size subsets, according to the codon 86 Gly or Val polymorphism; this adds an extra discrimination level to the typing. 108 samples were typed using the 1GA and the 2SA methods and no discrepancies were found. Typing indeterminations due to overlapping probe combinations were compared; it was found that the 2SA method, with the extra discrimination level at the PCR step, greatly improved resolution.

High Resolution Genetic Typing of the Class II HLA-DRB1 Locus Using Group-Specific Amplification and SSO-Hybridisation in Microplates

“…KOSTYU et al (1993) and GARCIA-PACHECO et al (1995) used a reversed system with an immobilized PCR product against which labeled probes were hybridized. Finally, PEPONNET et al (1995) used a more complicated strategy employing two PCR primer pairs, one for all DRBl alleles and one to distinguish the alleles based on codon 86 and subsequent typing of the products in microplates. Similar microplate typing systems have also been described for other HLA class I1 loci (BEIN et al 1994;GIORDA et al 1993).…”

“…1988). More recently, oligonucleotide probes have also been employed for typing of the DRBl locus using microplates, either by immobilizing the PCR product in the plate (KOSTYU et al 1993;GARCIA-PACHECO et al 1995) or by immobilizing the probes in the plate (CROS et al 1992;LAZARO et al 1993;KAWAI et al 1994;PEPONNET et al 1995).…”