Biotechniques

Doublex sequencing in molecular diagnosis of hereditary diseases.

Year 1998
Plaschke J. Voss H. Hahn M. Ansorge W. Schackert HK.
Technical University, Dresden, Germany.
We describe doublex sequencing of human genomic PCR products using two differently labeled primers in a single reaction and analysis on two automated DNA sequencing devices. Feasibility of the methodology is demonstrated by isothermal and cycle sequencing for two different PCR products and by cycle sequencing on both strands of a single product. It was applied to analyze mutations in patient DNAs in routine sample screening. Because it has the advantage of increased throughput and cost reduction while retaining its accuracy and reading length, we found that doublex sequencing is an attractive option for molecular diagnosis of hereditary diseases. This approach would be even more beneficial if it used DNA sequencing devices with several lasers in a single instrument.

Development of competitive mRNA PCR for the quantification of interleukin-6-responsive junB oncogene expression.

Year 1998
Igaz P. Fejer G. Szalai C. Toth S. Falus A.
Semmelweis University Medical School, Budapest, Hungary.
The transcription factor junB belongs to the jun family of protooncogenes. The appearance of junB mRNA in hepatic cells is an extremely early and sensitive marker of the action of proinflammatory cytokines including interleukin-6. In this study, a competitive reverse transcription (RT)-PCR assay has been developed that is suitable for the quantitative determination of junB mRNA expression. This nonisotopic assay compared to other methods (e.g., Northern blot) is a fast and convenient way to determine the expression of the junB gene and thus the immediate concentration- and time-dependent action of interleukin-6. Because interleukin-6 and interleukin-6-type cytokines play a highly important regulatory role in various pathophysiologically important processes, such as hepatic acute-phase reaction, the quantitative assay of junB mRNA completes the scale of laboratory approaches in inflammation and among other pathological conditions.