ABSTRACT
We have established a ligation based typing method to detect HLA-B*27 alleles at the DNA level. The method requires amplification of exon 2 of the HLA-B locus from genomic DNA by the polymerase catalyzed chain reaction (PCR) using group specific primers. An aliquot of the PCR amplification product, heat stable ligase and a pair of oligonucleotide probes, designed to hybridize adjacently to HLA-B*27 specific sequences of the amplified DNA are subsequently thermocycled. If the probes are perfectly complementary they become ligated otherwise they stay separated. The ligation of probes can be detected through their different labels by an enzyme linked immunosorbent assay (ELISA). Ligation based detection of beta-actin sequences which have been co-amplified serves as positive control for each PCR reaction. We observed complete concordance when typing 76 HLA-B*27 positive and 107 HLA-B*27 negative individuals either by serology or by the ligation based approach. We conclude that ligation based typing is a reliable tool for the DNA based detection of HLA-B*27 alleles. The procedure allows automation to a large extent and should be easily applicable to the typing of other HLA-class I alleles.
Subject(s)
HLA-B27 Antigen/genetics , Molecular Biology/methods , Base Sequence/genetics , Genotype , Humans , Leukocytes, Mononuclear , Polymerase Chain Reaction/methodsSubject(s)
Alleles , Genes, MHC Class II , HLA-DR Antigens/genetics , Base Sequence , Bone Marrow Transplantation , Child , Female , HLA-DR Serological Subtypes , HLA-DRB1 Chains , Histocompatibility Testing , Humans , Male , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Nucleic Acid , Tissue Donors , TurkeyABSTRACT
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.
