PHOTOTYPING - COMPREHENSIVE DNA TYPING FOR HLA-A, HLA-B, HLA-C, DRB1,DRB3, DRB4, DRB5 AND DQB1 BY PCR WITH 144 PRIMER MIXES UTILIZING SEQUENCE-SPECIFIC PRIMERS (PCR-SSP)

We have developed a single DNA typing method which uses 144 sequence-s pecific primer (SSP) reactions to simultaneously detect all known HLA- A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 specificities in an allele sp ecific or group specific manner using the same method, reagents, PCR p arameters and protocols for all loci. The results from this integrated class I & II method can be visualized on a single photographic or ele ctronic image and hence is described as ''Phototyping''. Phototyping h as an overall resolution greater than or equivalent to good serology a nd results can be obtained in under 3 hours making the method suitable for genotyping potential cadaver donor peripheral blood without serol ogical backup. This in turn produces the potential for reducing cold i schaemia times in renal transplantation as well as the application of prospective matching to cardiac and liver transplantation. The method has capacity to detect new alleles, for example, novel amplification p atterns suggestive of 4 new HLA-B alleles have been detected. The Phot otyping set has been used as the sole method of HLA typing for over 10 10 individuals. Phototyping is not problem-free; deviations from the s tandard protocol, poor quality DNA and unsuitable PCR machines can res ult in individual PCR failures or in incorrect assignment of antigens. Approximately 5% of genotypes were repeated (either partially or full y) because of incomplete or equivocal results.