GENOTYPE-RELATED FINGERPRINTS FROM HLA-DPB1 EXON-2 LOW-STRINGENCY PCR

Techniques based on the formation of heteroduplexes between relevant h eterologous amplified HLA loci have proved to be simple and cost-effec tive screening methods for the detection of DNA sequence diversity. Ho wever, the banding patterns produced may not be as complex as required . We used the original procedure of Pena et al. (Proceedings of the Na tional Academy of Sciences USA 91, 1946-1949, 1994) to generate finger prints from a specific, polymorphic PCR product. HLA-DPB1 exon 2 was a mplified, recovered from agarose gel, and used as a template for subse quent low-stringency (30 degrees C) amplification cycles (LS-PCR) in t he presence of a single primer. The LS-PCR products were run on 8% PAC E and silver-stained. In total, 22 subjects were characterized by this method. The issues of the reproducibility and specificity of the patt erns obtained were addressed by comparing fingerprints from individual s with the same genotype. The results showed that LS-PCR was robust. A further step was the evaluation of the diversity that can be generate d, i.e. the sensitivity of the method. Genotype-related fingerprints w ere produced, and differences as small as a single nucleotide in heter ozygous samples could be detected. We then demonstrated the usefulness of LS-PCR in the evaluation of donor/recipient pairs. We believe that LS-PCR may be a valuable adjunct to the battery of tests aimed at the verification of HLA matching before unrelated bone marrow transplanta tion. We suggest that it could be used to speed up the search process when several candidate donors are retrieved from registries before emb arking on SSOP typing or sequencing.