Use of nonisotopic M13 probes for genetic analysis: application to HLA class II loci

Previously, DNA polymorphisms in the HLA gene cluster have been analyzed using radioactive probes in Southern blot experiments; the restriction fragment length polymorphisms (RFLPs) revealed by this analysis are capable of subdividing HLA serological types. Here, we report the use of DNA probes labeled with biotinylated psoralen to provide nonisotopic detection of HLA class II RFLP patterns. These biotinylated probes contain cDNA sequences encoding the alpha and beta chains of DP, DQ, and DR HLA class II genes as inserts in M13 vectors. The recombinant M13 molecules are partially double-stranded with single-stranded HLA cDNA regions and contain biotinylated psoralen covalently linked to duplex DNA by UV irradiation. Following hybridization, the presence of biotinylated probe bound to target DNA is detected using a streptavidin-horseradish peroxidase conjugate, which converts the colorless substrate 3,3',5,5'-tetramethylbenzidine to a blue precipitate in less than 1 hr. The probe and detection system described here can detect single-copy genes in less than 0.5 microgram of total human DNA on Southern blots and generates the same specific RFLP patterns as do probes labeled with 32P by nick-translation. These biotinylated HLA class II probes have been applied to tissue typing for bone marrow transplantation and the study of insulin-dependent diabetes susceptibility, revealing in each case relevant polymorphisms not detected by serologic typing.