Quantitative detection of HIV-1 drug resistance mutations by automated DNA sequencing.

A comparison has been made between manual and automated DNA sequencing procedures to evaluate the ability to distinguish mixtures of wild-type and mutant sequences. Quantitative detection of such mixtures of HIV-1 drug resistance mutations was best achieved using an automated system that uses fluorescent-labelled sequencing primers. This procedure has a wide range of applications in clinical research, including heterozygote analysis. Software that automatically reports mixed-base positions is presented.

Isolation of plant nuclei.

The isolation of plant nuclei is a useful first step in many experiments concerned with the mechanism and control of gene expression in plants. For example, isolated nuclei can be used for the isolation of nuclear components such as chromosomal proteins (1), for the study of the processing of primary transcripts (2,3), for the assay and characterization of RNA polymerase activities (4-7), or for the measurement of transcription rates of specific genes (8) (see Chapter 37 ). A method is described here for the isolation of a crude preparation of intact plant nuclei, with an additional protocol for nuclei purification on a discontinuous gradient of Percoll (9) (see Note 1 in section 4). Centrifugation of crude nuclei preparations through Percoll gradients removes much of the contaminating cytoplasmic material such as starch grains (10), and the Percoll step appears to reduce the ribonuclease activity associated with nuclei (10). It is therefore recommended for transcription experiments. The crude nuclei preparation alone may be adequate for some work, for example, when attempting RNA polymerase assays for the first time, when very small amounts of tissue are involved, for preliminary experiments on the characterization of enzyme activities, or for the isolation of nuclear components that may be lost during purification.

In vitro transcription in plant nuclei.

Isolated plant nuclei can be used for fundamental studies on the transcription apparatus. Total RNA polymerase activity can be measured using plant nuclei, and by using different α-amanitin concentrations in the enzyme assay, the individual RNA polymerase I, II, and in activities can be measured (1-5). The assay procedure involves the incubation of nuclei in the presence of the four substrates for RNA synthesis: ATP, GTP, CTP, and UTP. If one of these precursors is supplied as a radio-labeled molecule, transcription can be detected as incorporation of radioactivity into acid-insoluble material. Following incubation, transcription products are precipitated with TCA, collected and washed on glass fiber filter discs, and counted by liquid scintillation counting.

Regional quality assessment of pH and blood gas analysers.

The performance of 41 pH and blood gas analysers in 20 hospitals was assessed using commercially available blood gas ampoules provided by seven manufacturers. The stability of the material and the effect of ambient temperature on Po2 was assessed. The overall mean values were outside the manufacturers' assigned values on eight out of 64 values. Using IL413 analysers as a basis for comparison, significant differences were found for Pco2 on ABL1, Corning 168 and 178 analysers and for Po2 on ABL1 and 2 and Corning 178. No significant differences were found for pH. Poor performers were identified in terms of imprecision. Analysers within or associated with clinical biochemistry departments gave better performance than those outside the laboratory. The five analysers that provided insufficient data for inclusion in the study were all situated outside the laboratory. Analysers from different manufacturers performed equally well provided they were used regularly and in accordance with manufacturers' instructions.