Nonisotopic M13 probes for detecting the beta-globin gene: application to diagnosis of sickle cell anemia.

Abstract

M13 DNA probes labeled with biotinylated psoralen and a streptavidin-horseradish peroxidase conjugate provide nonradioactive detection of the sickle cell and normal alleles of the beta-globin locus. The two biotinylated probes contain single-stranded sequences complementary to two different Sau3AI restriction fragments from the 5' region of the beta-globin gene and double-stranded M13 vector sequences. These probes are labeled with biotinylated psoralen photochemically linked to DNA. After hybridization, the presence of biotinylated probe bound to target DNA is detected in 3 h by using a streptavidin-horseradish peroxidase conjugate and the substrate, 3,3',5,5'-tetramethylbenzidine. Digestion of the normal (beta A) allele of the beta-globin gene with MstII (or isoschizomers) yields a 1.14-kb restriction fragment, while digestion of the mutant beta S allele yields a 1.34-kb fragment. These fragments can be resolved by gel electrophoresis and detected by Southern blot hybridization. The nonradioisotopic probe system can detect the beta-globin restriction fragment in as little as 0.5 microgram of human DNA and can distinguish heterozygotes (beta A beta S) from homozygotes (beta A beta A or beta S beta S) in 2.0 micrograms of human DNA.