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Jan 15, 2009 (Vol. 29, No. 2)

Enhancing PCR for Diagnostic Applications

Usage Ranges from the Functional Analysis of Genes to the Diagnosis of Disease

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    PCR, although discovered over two decades ago, continues to serve as a key topic of interest at numerous meetings, underscoring its importance in molecular biology. Two upcoming conferences previewed in this article, CHI’s “Molecular Medicine Tri-Conference” and the “4th International qPCR,” will focus on PCR and its specific application to molecular diagnostics.

    The technique uses a DNA polymerase to amplify or replicate DNA. As PCR progresses, the DNA generated is used as a template for further replication that sets a chain reaction in motion, generating millions of copies of the DNA piece. Real-time PCR is used to amplify and simultaneously quantify a targeted DNA molecule, adding immensely to the technique’s value in molecular biology in applications ranging from functional analysis of genes to the diagnosis of hereditary and infectious diseases.

    Sean Ferree, Ph.D., research and development manager at NanoString Technologies, will highlight his company’s recently introduced nCounter® Analysis System.

    “Using no enzymes or amplification, this novel system can analyze hundreds of genes in a single assay with high precision, even at low expression levels. We can assay directly from cell culture, tissue, or whole blood lysates,” he notes. “No sample purification is needed. We can also assay total purified RNA derived from formalin-fixed paraffin-embedded (FFPE) tissue samples.”

    Currently, the system is being used in custom gene-expression studies for microarray follow-up, biomarker detection, and pathogen detection using mRNA.  Off-the-shelf GPCR and kinase gene panels are under development. Dr. Ferree foresees that microRNA, copy-number variation, and direct protein analysis are additional future applications.

    The NanoString system uses digital technology that is based on direct multiplexed measurement of gene expression and can detect attomolar concentrations at less than one copy per cell. Molecular barcodes composed of four fluorescent colors and six spots are used as reporters to analyze the expression levels of up to 550 genes simultaneously. Single molecule imaging is then used to detect and count hundreds of unique transcripts in a single reaction. Because digital detection doesn’t rely on relative levels of fluorescence, only the order of fluors on the barcode strings need to be detected.

    Since the nCounter system requires no amplification of mRNA, the dynamic range necessary to measure the full range of expression in cells is simply the biological range of expression, typically three to four orders of magnitude, Dr. Ferree notes. Throughput is up to 72 samples in 24 hours, which at the 550-plex scale would correspond to 39,600 data points per day.  The entire assay takes 18 hours for one 12-sample cartridge with the 12-hour hybridization step typically being accomplished overnight, followed by two hours on the liquid-handling prep station and four hours on the digital analyzer. Since the latter two steps are automated, the total hands-on time for the user is less than 15 minutes.

    The system includes the automated prep station, a digital analyzer, the barcodes, and all reagents and consumables needed to perform the analysis.


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