SUMMARY Restriction endonuclease analysis of specific gene sequences is proving to be a valuable technique for characterisation and diagnosis of inherited disorders. This paper describes detailed protocols for isolation, restriction, and blot hybridisation of genomic DNA. Problems and alternatives in the procedure are discussed and a troubleshooting guide has been provided to help rectify faults.The development of techniques for the cloning1 and analysis2 of genes from complex organisms laid the foundation for the study of mutant genes associated with human inherited disorders. DNA from a person can now be cleaved into fragments of defined length by restriction endonucleases. The fragments are then separated by gel electrophoresis, blotted onto filters,2 and incubated with radioactively labelled gene specific probes. These probes, obtained by molecular cloning techniques, are isolated and characterised DNA sequences which will associate specifically with homologous genomic DNA sequences on the filter. Thus, only fragments containing part or all of the gene of interest will be detected. This new recombinant DNA technology was rapidly applied to the molecular characterisation and antenatal diagnosis of the haemoglobinopathies and thalassaemia.3-7 As cloning techniques have become more sophisticated, the number of purified, cloned human genes has proliferated to the extent that a recently published list8 is already out of date. A considerable number of genetic diseases are therefore amenable to DNA analysis, and the use of linked restriction fragment length polymorphisms9 10 has further extended the applicability of the technique.These advances have brought DNA analysis within the scope of the clinical geneticist, and the techniques will ultimately become part of the routine service provided by human genetics departments. DNA blotting and molecular hybridisation
A new alpha thalassemia defect has been detected in the South African population. Restriction mapping of the alpha globin gene cluster in affected individuals has established that the defect is associated with the removal of 22.8-23.7 kb of DNA, including the psi zeta 1, psi alpha 1, psi alpha 2, alpha 2 and alpha 1 globin genes. The 5' endpoint of the deletion has been localized between the zeta 2 and psi zeta 1 globin genes, and the 3' endpoint lies 4-5 kb 3' to the alpha 1 globin gene. We have called the deletion - -SA in order to distinguish it from alpha zero thalassaemia defects described in other populations.
This study examines the influence of cholera toxin (CT) on T lymphocyte activation by the mitogenic lectin phytohaemagglutinin (PHA). CT suppressed lectin-induced [3H]thymidine uptake in a dose-dependent fashion and acted synergistically with PHA in the generation of intracellular cyclic AMP. The toxin was assumed to act on Gs, because it also stimulated ADP-ribosylation of a 45 kDa membrane protein in vitro; no additional substrates were seen. The inhibitory effect of the adenylate cyclase/cyclic AMP pathway was shown to be directed at a concomitant stimulatory pathway, namely inositol phospholipid turnover. Lectin-stimulated 32P incorporation into both phosphatidylinositol as well as its 4,5-biphosphate derivative was depressed in the presence of CT or exogenous dibutyryl cyclic AMP. This, in turn, was associated with reduced activation of C-kinase as determined by decreased lectin-induced translocation from the cytosol to the surface membrane. These results indicate that Gs probably acts as a transducer between the PHA receptor and adenylate cyclase and may give rise to an exaggerated adenylate cyclase response in the presence of CT. It would seem as if reduction in inositol phospholipid turnover is related to the elevation of cyclic AMP rather than a CT effect on a putative transducer which acts directly on phospholipase C. Our study does not exclude the existence of non-CT-sensitive transducers in this capacity.
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