A Combination of Two Distinct in vitro Amplification Procedures for DNA Typing of HLA‐DRB and ‐DQB 1 Alleles

Abstract

The differential hybridisation of oligonucleotide probes to polymerase chain reaction (PCR)-amplified DNA has become a standard procedure for tissue typing. We describe a typing method in which differential ligation replaces differential hybridisation, which is a significant simplification of this strategy. After amplification by the PCR two labelled, sequence-specific oligonucleotides hybridise, in the fluid phase, to one strand of heat-denatured amplification product in juxtaposition. In the case of perfectly complementary sequences surrounding the gap, a thermostable ligase catalyses the ligation of the two oligonucleotides, otherwise they stay separated. The use of heat-resistant ligase enables easy repetition of the denaturation-annealing-ligation cycle in a thermocycler. The ligation products are detected by an enzyme linked immunosorbent assay. We tested this typing approach in a model system, the characterisation of three functional alleles of HLA-DRB3 using three probe pairs. No discrepancies were observed in typing 100 individuals of known genotypes. A total of 33 probe pairs combined with generic and group-specific amplification allowed the typing of alleles of HLA-DRB and -DQB1 loci at low resolution. We confirmed ligation-based typing results of 259 individuals with sequence-based HLA-DRB1 typing and HLA-DQB1 typing using PCR with sequence-specific primers (SSPs). In addition, more than 1,500 ligation-based HLA-DRB1 typings were concordant with SSP typing. Excellent signal-to-noise ratios in the enzyme-linked immunosorbent assay make ligation-based typing remarkably robust. The time requirement of 2.5 h post-PCR enables practicable typing of putative organ donors. The whole procedure is more easily amenable to automation than methods based on differential hybridisation requiring additional incubators and extra handling for hybridisation and washing.

Nitration of the Pollen Allergen Bet v 1.0101 Enhances the Presentation of Bet v 1-Derived Peptides by HLA-DR on Human Dendritic Cells

“…Low resolution analysis of the HLA-DRB3/4/5 tissue type was performed using a ligation based typing approach [43]. High resolution analysis of HLA-DRB1 and HLA-DRB3/4/5 tissue types was performed by nucleotide sequencing of exon 2 (BigDye Terminator Cycle Sequencing Kit, ABI, Foster City, CA).…”