Allele-specific hybridization using oligonucleotide probes of very high specific activity: discrimination of the human beta A- and beta S-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 beta-globin (beta A) or to the sickle cell human beta-globin (beta s) gene. Template-directed polymerization of highly radiolabeled alpha[32P]deoxyribonucleoside triphosphates (dNTPs) onto nonamer and decamer primers produced probes with specific activities ranging from 1.0 X 10(10) to 2.0 X 10(10) dpm/micrograms. The extremely high specific activities of these probes made it possible to detect the beta A and beta S single-copy gene sequences in as little as 1 microgram of total human genomic DNA as well as to discriminate between the homozygous and heterozygous states.