phosphorus-radioisotopes and Genetic-Diseases--Inborn

Recent developments in recombinant DNA technology have made possible the production of gene probes consisting of cloned gene segments, cloned segments of DNA linked to genes, and synthetic gene fragments. Several methods have been developed by which these probes may be used for the diagnosis of human disease. This technology has been outstandingly successful for prenatal diagnosis and carrier detection in many genetic diseases. These methods have also been successfully applied to the analysis of human malignancies, by providing for the determination of cell lineage and clonality in lymphoid neoplasms. Finally, these methods have shown potential for rapid and sensitive diagnosis of some infectious diseases.

Topics: B-Lymphocytes; Bacterial Infections; Biotin; Cloning, Molecular; Deoxyribonucleotides; DNA; DNA Restriction Enzymes; DNA, Recombinant; Female; Genetic Carrier Screening; Genetic Diseases, Inborn; Genotype; Hemophilia A; Humans; Immunoglobulins; Infections; Leukemia, Lymphoid; Leukocytes; Male; Muscular Dystrophies; Nucleic Acid Hybridization; Phenylketonurias; Phosphorus Radioisotopes; Polymorphism, Genetic; Pregnancy; Prenatal Diagnosis; Receptors, Antigen, T-Cell; RNA; T-Lymphocytes; Virus Diseases

Topics: Adenosine Triphosphate; Base Sequence; DNA Primers; DNA, Mitochondrial; Genetic Diseases, Inborn; Humans; Kearns-Sayre Syndrome; Molecular Sequence Data; Mutation; Ophthalmoplegia; Phosphorus Radioisotopes; Polymerase Chain Reaction; Repetitive Sequences, Nucleic Acid; Sequence Deletion

The seemingly autonomous amplification of naturally occurring triplet repeat sequences in the human genome has been implicated in the causation of human genetic disease, such as the fragile X (Martin-Bell) syndrome, myotonic dystrophy (Curshmann-Steinert), spinal and bulbar muscular atrophy (Kennedy's disease) and Huntington's disease. The molecular mechanisms underlying these triplet amplifications are still unknown. We demonstrate here that a synthetic (CGG)17 oligodeoxyribonucleotide can be utilized as hybridization probe to visualize some of the triplet repeats in the human genome. This technique may help in studies aimed at the elucidation of the amplification mechanism.

Topics: Autoradiography; Base Sequence; Blotting, Southern; DNA; Genetic Diseases, Inborn; Genome, Human; Humans; Molecular Sequence Data; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Phosphorus Radioisotopes; Repetitive Sequences, Nucleic Acid; Restriction Mapping

Polymerase chain reaction, using thermostable DNA polymerase, has revolutionized DNA diagnostics. Another thermostable enzyme, DNA ligase, is harnessed in the assay reported here that both amplifies DNA and discriminates a single-base substitution. This cloned enzyme specifically links two adjacent oligonucleotides when hybridized at 65 degrees C to a complementary target only when the nucleotides are perfectly base-paired at the junction. Oligonucleotide products are exponentially amplified by thermal cycling of the ligation reaction in the presence of a second set of adjacent oligonucleotides, complementary to the first set and the target. A single-base mismatch prevents ligation/amplification and is thus distinguished. This method was exploited to detect 200 target molecules as well as to discriminate between normal beta A- and sickle beta S- globin genotypes from 10-microliters blood samples.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA Ligases; Escherichia coli; Gene Library; Genetic Diseases, Inborn; Globins; Humans; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Oligonucleotide Probes; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Thermus