HLA Class II typing by digestion of PCR‐amplified DNA with allele‐specific restriction endonucleases will fail to unequivocally identify the genotypes of many homozygous and heterozygous individuals

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Abstract
Recently, a new technique for HLA class II genotyping has been introduced, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, claimed to be a practical alternative to conventional serological and cellular HLA class II typing (1-3). The PCR-RFLP technique is ingenious, relatively rapid and does not require hybridization with sequence-specific oligonucleotide probes. However, analysis of whether homozygous and heterozygous combinations of PCR-RFLP patterns for the various investigated HLA class II loci are unique or not unfortunately shows that only 19% of DRB homozygous and heterozygous combinations are unique. The figures for the DQA1, DQB and DPB loci are 56%, 29% and 65%, respectively. As not all nucleotide sequences analyzed in the above-mentioned studies (1-3) gave rise to unique PCR-RFLPs and as more sequences new are known, for the DRB1, DRB3, DRB5 and DPB1 loci (4), the frequencies of unique PCR-RFLP patterns for the different HLA class II loci will be reduced even further. Thus, the present analysis demonstrates that the PCR-RFLP technique, performed as described in references 1-3, is not yet ready to be used for routine HLA class II genotyping. The resolution of the PCR-RFLP method can be improved by various modifications. However, the role of a modified PCR-RFLP technique in HLA class II typing still remains to be shown.