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Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.

The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. We have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. We also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single 1)NA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.Inactivation of the human hypoxanthine phosphoribosyltransferase (HPRT) gene (1) leads to the Lesch-Nyhan syndrome (LN), an X chromosome-linked, genetically lethal, neurological disease (2). Partial HPRT activity results in a severe form of gouty arthritis (3). Both conditions are characterized by elevated levels of uric acid in serum, and LN is also associated with choreoathetosis, spasticity, mental retardation, and a bizarre form of self-mutilation.Approximately 15% of LN patients have major gene rearrangements that can be detected by Southern analysis (4). The majority of cases have normal size and amounts of HPRT mRNA when analyzed by Northern analysis (4,5), but :35% of these reveal molecular lesions when assayed by ribonuclease digestion of RNARNA heteroduplexes (6). Only a small percentage produce detectable HPRT protein (5). Overall, these data indicate a heterogeneity of mutations in the disease, with a preponderance of single DNA base changes. In combination with a paucity of informative polymorphic DNA markers at the HPRT locus, the mutational heterogeneity has considerably hampered the application of DNA-based techniques to LN family analysis.Recent developments in DNA technology have greatly increased the simplicity and speed with which single base changes in human DNA can be identified. In particular, the development of the polymerase chain reaction (PCR) (7-9) has provided a procedure to enrich for a specific cDNA so that DNA sequence analyses may be performed without the need for time-consuming library construction and screening (10). Fluorescently labeled DNA oligonucleotides have been combined with dideoxynucleotide termination DNA sequencing and real-time laser gel scanners to automate DNA sequencing of cloned materials (11). When a DNA sequence change is identified, the mutant allele can be detected by differential hybridization to allele-specific oligonucleotides (ASOs) (12). We have adapted and extended these procedures to detect LN mutations by automated direct DNA sequencing of PCR-amplified HPRT cDNA. These strategies now permit simplified diagnoses of the mutations in the majority of LN affected families. Oligonucleotides. Oligonucle...

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