Direct Detection of HIV-1 RNA from AIDS and ARC Patient Samples

Murakawa, George J.; Zaia, John A.; Spallone, Patricia; Stephens, Delilah A.; Kaplan, Bruce; Rb, Wallace; Rossi, John J.

doi:10.1089/dna.1988.7.287

Cited by 117 publications

(30 citation statements)

References 28 publications

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“…Our results are in support of the potential value of RT-PCR as a simple and specific method for the detection of plant RNA viruses in infected sap. Similar tests using a reverse transcription step prior to cDNA amplification have been developed for detection of viroids (Puchta et al, 1989;Hadidi et al, 1990), for several animal RNA viruses including rhinovirus (Gama et al, 1989), HIV (Byrne et al, 1988;Hart et al, 1988;Murakawa et al, 1988), human picornaviruses (Hyypia et al, 1989), and for plant RNA viruses (Vunsh et al, 1990;Korschineck et al, 1991). These reports describe methods that use a previous step for RNA purification.…”

Section: Discussionmentioning

confidence: 99%

An appraisal of different methods for the detection of the walnut strain of cherry leafroll virus

Borja¹,

Ponz²

1992

Journal of Virological Methods

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Three methods were evaluated for the detection of cherry leafroll virus: ELISA, dot-blot and reverse transcriptional polymerase chain reaction (RT-PCR). Dot-blot and RT-PCR were carried out in crude plant extracts without any further RNA purification. Dot-blot hybridization using a 32P-labelled DNA probe was as sensitive as previously reported ELISA results for cherry leafroll virus detection. The most sensitive method was RT-PCR, which amplified a specific fragment of 448 bp from the 3' untranslated region of both viral genomic RNAs. RT-PCR was used to detect cherry leafroll virus in infected walnut buds and twigs.

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Section: Discussionmentioning

confidence: 99%

An appraisal of different methods for the detection of the walnut strain of cherry leafroll virus

Borja¹,

Ponz²

1992

Journal of Virological Methods

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“…The early detection of such resistance could hasten the development of alternative clinical protocols. Recently, enzymatic amplification methods have been developed to detect genes associated with human diseases (3,4). This assay, known as the polymerase chain reaction (PCR), has been modified to amplify a sequence of a drug resistance gene with two flanking oligonucleotides acting as primers for DNA synthesis ( Fig.…”

Section: Introductionmentioning

confidence: 99%

Utility of the polymerase chain reaction in detection of gene expression in drug‐resistant human tumors

Scanlon

Kashani‐Sabet

1989

Clinical Laboratory Analysis

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Recently, an enzymatic amplification method, the polymerase chain reaction (PCR), was modified to amplify a sequence of a drug resistance gene. The PCR assay can confirm data achieved by conventional molecular biology techniques, while requiring less time and fewer patient cells. It can be quantitated for gene expression. The data generated make it possible to analyze m-RNA expression in tumor samples without being limited to detecting only gene amplification in response to cancer chemotherapy. The PCR assay can be an effective device in the early detection of resistance to chemotherapy.

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“…Gel electrophoresis, transfer to a nylon filter, hybridization, and washing were as described (31). The radioactivity of each band on the filter was determined by an Ambis radioisotope scanning system II (Automated Microbiology Systems, San Diego).…”

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confidence: 99%

A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells.

Singer-Sam

Goldstein

Dai

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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Hpa II site H8 is in the CpG-rich 5' untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGKI). It is the only Hpa II site in the CpG "island" whose methylation pattern is perfectly correlated with transcriptional silence of this gene. We measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. We conclude that site H8 is unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.Female primordial germ cells of mammals resemble somatic cells in having both an active and an inactive X chromosome. Evidence for inactivity of one X chromosome of oogonia has been found both cytologically and by studies on glucose-6-phosphate dehydrogenase heterodimer formation (1, 2). After migration of the oogonia to the genital ridge at, or shortly before, the time of entry into meiosis, reactivation of the previously inactive X chromosome occurs (1-6). DNA methylation has been correlated with X chromosome inactivation in many studies (reviewed in refs. 7-9) and it is of interest to determine whether a loss of methylation is concomitant with X chromosome reactivation during oogenesis. Data on DNA methylation of oogenic cells are sparse. Several years ago evidence was obtained for undermethylation of some repeated sequences in female germ-line DNA (10, 11), and more recently, Driscoll and Migeon (12) obtained evidence for lack of methylation of several X-linked and autosomal genes in oogenic and spermatogenic cell fractions.As the latter study was done on tissue fractions rather than preparations free of somatic cells, we thought it was important to measure DNA methylation of highly purified germ cells. As such cells are isc. table only in small quantities, we used a polymerase chain raction (PCR)-based assay previously used to measure DNA methylation at a CCGG site in individual mouse embryos (13).The site we chose to assay was Hpa II site H8 in the 5' untranslated coding region of the human X-linked PGKI gene, 20 base pairs (bp) downstream of the major transcription start site (14). The 5' region of the PGKI gene is now the most highly characterized X-linked promoter region, and the relationship between methylation, transcription, and transcription factors has been studied both in normal human lymphocytes and in cultured cell lines (15)(16)(17)(18)(19). Recently, ligation-mediated genomic sequencing has been used to establish that the promoter region of the PGKI gene on the inactive X chromosome is methylated at 60 of 61 CpG sites, whereas the active promoter has no methylated sites (19). Prior to these studies, methylation-sensitive restriction enzymes had been used to investigate methylation at the 8 Hpa II sites in the PGKI 5' region in lymphocytes (15) and in cell lines before and after...

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Direct Detection of HIV-1 RNA from AIDS and ARC Patient Samples

Cited by 117 publications

References 28 publications

An appraisal of different methods for the detection of the walnut strain of cherry leafroll virus

An appraisal of different methods for the detection of the walnut strain of cherry leafroll virus

Utility of the polymerase chain reaction in detection of gene expression in drug‐resistant human tumors

A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells.

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