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Biomedical subjects

R G Brownlee

Publications and source records attributed to R G Brownlee.

7 recordsLinked to original sources
PubMed1991

Analysis of DNA restriction fragments and polymerase chain reaction products towards detection of the AIDS (HIV-1) virus in blood.

A high-performance capillary electrophoresis system with a polysiloxane-coated capillary and polymeric buffer additives was investigated for the analysis of DNA restriction fragments and polymerase chain reaction (PCR) products. Mobility data and Ferguson plots of the DNA fragments at different polymer (hydroxypropylmethylcellulose) concentrations indicated that effective molecular sieving was obtained consistent with existing data of conventional gel electrophoresis and with recent HPCE data. The precision and peak efficiency were excellent and the system was applied to the analysis of specific co-amplified DNA sequences (HIV-1 and HLA-DQ-alpha). After PCR, ultrafiltration was used in the sample preparation step to desalt the sample and to remove superfluous PCR reaction products. Electrokinetic injection was used for sample introduction into the capillary. The addition of ethidium bromide to the buffer resulted in longer migration times of DNA fragments and better peak resolution. During HPCE, an artifact associated with dilute DNA solutions leading to the appearance of extra peaks in the electropherogram was found.

Blood↗
PubMed1991

Rapid simultaneous detection of multiple retroviral DNA sequences using the polymerase chain reaction and capillary DNA chromatography.

The polymerase chain reaction (PCR) technique is a powerful new tool for amplifying target DNA, thus allowing for sensitive detection of specific nucleic acid sequences. One important potential use of PCR involves screening the donated blood supply for transfusion-transmitted viruses. Realization of this goal has been limited by (1) the requirement for multiple, discrete PCR reactions to amplify and detect target sequences of more than one virus, and (2) the lack of a rapid, nonhazardous means for specific detection of one or more PCR-amplified products. We report the simultaneous amplification of three distinct target sequences without discernable loss in sensitivity toward any single target sequence. We also demonstrate very rapid separation and detection of PCR-amplified viral DNA through the use of automated capillary DNA chromatography. Amplified DNA peaks were initially identified by scanning the capillary effluent at ultraviolet wavelengths, while discrimination of human immunodeficiency virus type 1 and human T-cell leukemic virus type I PCR-amplified DNA was accomplished through use of virus-specific, fluorescently labeled primers and probes. These results indicate progress toward an automated system for screening the blood supply for nucleic acid sequences of multiple pathogens.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Application of capillary DNA chromatography to detect AIDS virus (HIV-1) DNA in blood.

The application of capillary DNA chromatography to the detection of the AIDS human immunodeficiency virus, type 1 (HIV-1) proviral DNA in blood is reported for the first time. Combining polymerase chain reaction with fluorescence-labeled DNA probes provides the basis for the amplification and specific detection of the DNA. Samples analyzed for HIV-1 DNA included both infected human blood and cell cultures. The new DNA separation method, capillary DNA chromatography (related to both capillary electrophoresis and capillary hydrodynamic chromatography), is shown to be a powerful method of analysis for DNA. These preliminary results indicate that an automated approach to screening the blood supply for HIV-1 may become a reality in the future.

Chromatography↗
PubMed1988

Separation and detection of DNA by capillary electrophoresis.

The use of capillary electrophoresis for the separation and detection of nucleic acids has been investigated. Lab-model instruments have been built, using commercially available UV absorbance and fluorescence detectors which were modified for use with 50-100 microns I.D. fused-silica capillary tubing. The sensitivity of these instruments (signal-to-noise ratio = 3) was measured as 15 micrograms/ml for fluorescence detection of ethidium bromide-stained herring-sperm DNA and 3 micrograms/ml for UV absorbance detection. With the former instruments a variety of strategies has been used to attain rapid separations of bases, oligonucleotides, restriction fragments and whole phage, viral and plasmid DNAs.

DNA↗
PubMed1988

Capillary electrophoresis.

While capillary electrophoresis, or historically related techniques, have been used for over a century, and recognition of the value of this separation methodology has certainly grown rapidly in the past few years, the technique has generally been used by analytical chemists, particularly in Europe and Japan, and small groups of researchers in the United States. Many of the basic instrumentation problems have been solved only relatively recently, and researchers using capillary electrophoresis are now turning their attention to studying specific applications which demonstrate the potential versatility of this electrophoretic technique. The appearance of standardized commercial instrumentation is imminent. With the availability of such technology, capillary electrophoresis will no longer be an academic curiosity, but rather a tool with the potential for routine separations of diverse samples of interest to analyst, researcher, and clinician.

Electrophoresis↗