Allele-Specific Hybridization Using Oligonucleotide Probes of Very High Specific Activity: Discrimination of the Human βA- and βS-Globin Genes

Abstract

The repair activity of Escherichia coli DNA polymerase I (Klenow fragment) was used to prepare nonadecanucleotide hybridization probes which were complementary either to the normal human β-globin (β^A) or to the sickle cell human β-globin (β^S) gene. Template-directed polymerization of highly radiolabeled α[³²P]deoxyribonucleoside triphosphates (dNTPs) onto nonamer and decamer primers produced probes with specific activities ranging from 1.0 × 10¹⁰ to 2.0 × 10¹⁰ dpm/μg. The extremely high specific activities of these probes made it possible to detect the β^A and β^S single-copy gene sequences in as little as 1 μg of total human genomic DNA as well as to discriminate between the homozygous and heterozygous states.