Brenda J. Conner

Two 19-base-long oligonucleotides were synthesized, one complementary to the normal human beta-globin gene (beta A) and one complementary to the sickle cell beta-globin gene (beta S). The nonadecanucleotides were radioactively labeled and used as probes in DNA hybridization. Under appropriate hybridization conditions, these probes can be used to distinguish the beta A gene from the beta S allele. The DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) only hybridized with the beta A specific probe; the DNA from those homozygous for the sickle cell beta-globin gene (beta S beta S) only hybridized with the beta S specific probe. The DNA from heterozygous individuals (beta A beta S) hybridized with both probes. This allele-specific hybridization behavior of oligonucleotides provides a general method for diagnosis of any genetic disease which involves a point mutation in the DNA sequence of a single-copy gene.

We investigated a method employing synthetic oligonucleotides for the prenatal diagnosis of beta-thalassemia due to a single nucleotide mutation. The beta 0 thalassemia we tested is produced by a nonsense mutation and is commonly found in Sardinia and other parts of the Mediterranean. In this DNA lesion, the glutamine codon CAG at the beta 39 position is mutated to TAG, which results in a stop codon and premature termination of the beta-globin chain. We synthesized two oligonucleotides: one homologous to the normal beta A gene and the other to the beta 0 thalassemia gene at the beta 39 location. The oligonucleotides were labeled with 32P and used as hybridization probes for normal and thalassemic DNA. The beta A probe hybridized only to the normal DNA, and the beta-thalassemia probe only to thalassemic DNA, thus providing a technique for direct demonstration of the mutation. The method is sensitive enough to be applied directly to DNA that is isolated from uncultured cells obtained from only 20 ml of amniotic fluid as early as the 16th gestational week.

Synthetic nonadecanucleotides complementary to the human beta A-, beta S-, or beta C-globin sequences were used as hybridization probes to screen human genomic DNA samples for these genes. The oligonucleotides were 32P-labeled and used as probes to genotype restriction endonuclease digests of human genomic DNA. The data obtained show that hybridization with oligonucleotide probes, unlike restriction fragment length polymorphism (RFLP) analysis or direct restriction enzyme digestion, can be used to directly distinguish among the three alleles of beta-globin, beta A, beta S, and beta C, when present either in one (heterozygous) or two copies.

Using oligonucleotide hybridization, single and multiple nucleotide differences between alleles were detected directly in genomic DNA without electrophoretic separation. The DNA was immobilized in depressions in an agarose gel (in situ dots) and hybridized with radiolabeled, allele-specific oligonucleotide probes. An oligonucleotide complementary to a unique sequence region of the human major histocompatibility complex gene HLA-B27 only hybridized with genomic DNA from an HLA-B27-positive individual. Two other oligonucleotides complementary to the normal human beta-globin gene (beta A) and to the sickle cell globin gene (beta S) were synthesized. Using competition hybridization conditions which included the presence of a 10-fold molar excess of unlabeled oligonucleotide complementary to the other beta-globin allele, DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) hybridized to the beta A probe exclusively, whereas DNA from individuals homozygous for the sickle cell globin gene (beta S beta S) hybridized only with the probe for the sickle cell gene. As expected, DNA from heterozygous individuals bound to both probes. Similar results were obtained with total human RNA immobilized in in situ dots. Possible applications of this methodology include genetic disease diagnosis, population carrier screening, HLA "DNA" typing, and DNA and RNA sequence polymorphism analysis.