In solutions of tetraalkylammonium salts the melting temperature of oligonucleotide duplexes is independent of nucleotide sequence and thus GC content. Data quantitating the destabilizing effects of various mismatches in these solvents are also presented. The results are in accord with theories on DNA melting and establish conditions under which oligonucleotides can be used as hybridization probes with predictable and controllable specificity.

A novel approach for the detection of specific DNA or RNA sequences based on branch migration and DNA strand displacement is described. A partially double-stranded probe complex is prepared with a detectable label on one of the two strands and incubated with analyte molecules under hybridization conditions. The analyte molecules hybridize to the single-stranded portion of the probe complex and undergo branch migration to release the labelled DNA strand from the complex. Initial characterization of the assay indicates that both qualitative and quantitative information about analytes present in a sample can be obtained. The strand displacement assay is more sensitive to sequence alterations in the analyte than is a hybridization assay and can be promoted by rec A protein at 37 °C. Finally, a method for preparing probe complexes by cloning in a single-stranded DNA vector is also described.

This novel method for the detection of specific nucleic acid sequences has potential applications to clinical diagnosis. During hybridization, a signal-bearing nucleic acid strand is displaced by the target nucleic acid from a partially single-stranded complementary probe strand of nucleic acid. The probe:signal strand complex is prepared by hybridizing single-stranded probe that is entirely complementary to the target nucleic acid with a shorter signal sequence that is complementary to a portion of the probe strand. The sample nucleic acid is added to this hybrid complex under hybridization conditions. The target sequence, if present in the sample, will hybridize first to the unoccupied probe sequences, and then will displace the labeled strand by branch migration. By this "strand displacement" the signal strands are freed in solution, where they may be separated from those still hybridized; the quantity of label measured is directly proportional to the amount of analyte sequences in the sample. This method, demonstrated here for model and synthetic DNAs, can easily be adapted for the detection of any RNA or DNA sequence and obviates the need for immobilization of sample. A wide variety of labeling techniques can be used, and the displacement can be performed in solution or with the hybrid complex attached to a solid support. This assay circumvents nonspecific binding of label to the filter matrix and the laborious washing steps inherent in other assays involving nucleic acid probes.

The glycoprotein hormone erythropoietin regulates the level of oxygen in the blood by modulating the number of circulating erythrocytes, and is produced in the kidney or liver of adult and the liver of fetal or neonatal mammals. Neither the precise cell types that produce erythropoietin nor the mechanisms by which the same or different cells measure the circulating oxygen concentration and consequently regulate erythropoietin production are known. Cells responsive to erythropoietin have been identified in the adult bone marrow, fetal liver or adult spleen. In cultures of erythropoietic progenitors, erythropoietin stimulates proliferation and differentiation to more mature red blood cells. Detailed molecular studies have been hampered, however, by the impurity and heterogeneity of target cell populations and the difficulty of obtaining significant quantities of the purified hormone. Highly purified erythropoietin may be useful in the treatment of various forms of anaemia, particularly in chronic renal failure. Here we describe the cloning of the human erythropoietin gene and the expression of an erythropoietin cDNA clone in a transient mammalian expression system to yield a secreted product with biological activity.

Mammalian α- and β-like globin polypeptides are encoded by a small family of genes that are differentially expressed during development and, in some species, during adult erythroid cell maturation (Kitchen and Brett 1974; Bunn et al. 1977). Our laboratory has been studying the structure, evolution, and chromosomal arrangement of globin genes in two mammalian species, human and rabbit.

Deletions in the DNA of individuals with hereditary persistence of fetal haemoglobin (HPFH) and 8 beta-thalassaemia have been mapped as a means of identifying regulatory sequences involved in the switch from fetal to adult globin gene expression. The end points of these deletions have been precisely located with respect to restriction endonuclease cleavage sites within and surrounding the gamma-, delta- and beta-globin genes in normal human DNA and the deletion maps were used to obtain definitive evidence for the physical linkage of the fetal and adult beta-like globin genes in the order 5'Ggamma-Agamma-delta-beta 3'. Correlation of haematological data and the location of deletions in two cases of HPFH and one case of deltabeta-thalassaemia suggest that a region of DNA located near the 5'-end of the delta-globin gene may be involved in the suppression in cis of gamma-globin gene expression in adults. The interpretation of a second case of deltabeta-thalassaemia is complicated by the fact that the deletion removes the Agamma-gene in addition to the region near the 5'-end of the delta-globin gene.