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摘要
A simple, rapid, and precise method of typing HLA class II polymorphism would be valuable in the areas of disease susceptibility, tissue transplantation, individual identification, and anthropological genetics. Herein, we describe a method of analyzing class II sequence polymorphism based on polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes. Many more DNA-defined alleles at the class II loci have been identified than can be distinguished by conventional serologic typing. Consequently, matching transplant donors and recipients by PCR/oligonucleotide typing should reduce graft rejection and graft-vs-host disease. Also, the ability to identify alleles conferring genetic predisposition to specific diseases (eg, insulin-dependent diabetes mellitus) is significantly enhanced by distinguishing the many alleles or ''subtypes'' within a serologic type (eg, DR4). One valuable property of sequence-based HLA typing strategies, like oligonucleotide probe hybridization, is that they reveal how and where two alleles differ, not simply that they can be operationally distinguished. The nature and location of HLA polymorphisms appears to be critical in disease association studies and are important in tissue typing for transplantation. New alleles at the DRB1, DPB1, and DQB1 loci are likely to be identified as this technology is applied to more and more samples, particularly in nonwhite ethnic groups. A new allele is uncovered as an unusual pattern of probe binding and then confirmed by sequencing. This pattern is observed because class II polymorphism is localized to specific regions and virtually all ''new'' alleles represent ''shuffled'' combinations of polymorphic sequences found in previously known alleles. Since these polymorphisms are in the region of probe binding, these new alleles can be detected without increasing the probe panel. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available would not be revealed by oligonucleotide typing. With the PCR primers and probes described here, 7 DQalpha1 alleles, 15 DQbeta1 alleles, 18 DPB1 alleles, and 32 DRB1 alleles are distinguished. Additional primers and/or probes can, of course, increase the allelic discrimination of PCR/oligonucleotide probe typing. These horseradish peroxidase-labeled oligonucleotide probes are stable (>2 years when stored at 4-degrees-C) and the typing system is simple and robust. Although this dot blot/oligonucleotide hybridization procedure is a powerful and precise method of HLA class II typing, the complexity of the procedure increases as the number of probes required for analysis increases. The reverse dot blot method, based on an array of immobilized probes, allows the typing of individual samples in one single hybridization reaction. In this approach, a panel of unlabeled oligonucleotides are immobilized to a nylon membrane. The PCR product is labeled during the amplification reaction by using biotinylated primers and hybridized to the membrane. The presence of bound PCR product specifically hybridized to a given probe is detected using a streptavidin-horseradish-peroxidase conjugate and either chromogenic or chemoluminescent substrates. This method, which has also been applied to the detection of beta-thalassemia and cystic fibrosis mutations, offers the simplest and most rapid approach to the HLA typing of clinical samples.
- 出版日期1993-5
