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Clinical Chemistry 51: 1523-1525, 2005; 10.1373/clinchem.2005.049197
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(Clinical Chemistry. 2005;51:1523-1525.)
© 2005 American Association for Clinical Chemistry, Inc.

Technical Briefs

LightTyper Assay with Locked-Nucleic-Acid–Modified Oligomers for Genotyping of the Toll-Like Receptor 4 Polymorphisms A896G and C1196T

Silke Grannemann1, Olfert Landt2, Sebastian Breuer3 and Brunhilde Blömeke3,a

1 Department of Dermatology and Allergology, University Hospital of the RWTH Aachen, Aachen, Germany;
2 TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany;
3 Department of Ecotoxicology/Toxicology, University Trier, Trier, Germany;

aaddress correspondence to this author at: Department of Ecotoxicology/Toxicology, University Trier, Am Wissenschaftspark 25-27, 54296 Trier, Germany; fax 49-651-201-3780, e-mail bloemeke@uni-trier.de

The first 300 words of the full text of this article appear below.

The toll-like receptor-4 (TLR4; OMIM*603030), a member of a large family of transmembrane proteins, is predominantly expressed on monocytes and macrophages (1). TLR4 is involved in various diseases and in innate and adaptive immunity (2), and it is thought to be crucial in mediating lipopolysaccharide effects (3). The common co-segregating variants, Asp299Gly and Thr399Ile, affect the extracellular domain of the TLR4 receptor. These variants are associated in humans with a blunted response to inhaled lipopolysaccharide in humans and lead to an altered host immune response to pathogens (4).

The corresponding TLR4 genetic variations A896G (D299G; OMIM*603030.001) and C1196T (T399I, OMIM*603030.002) have been analyzed by use of DNA sequencing (4), restriction fragment length polymorphism analysis (5), MGB-TaqMan probes, matrix-assisted laser desorption/ionization analysis (6), and LightCycler hybridization probes (7). We developed a new homogeneous assay for genotyping of A896G and C1196T by use of locked-nucleic-acid (LNA)–modified SimpleProbe oligomers (8) on the LightTyper instrument (Roche). This assay provides a specific and sensitive method for high-throughput genotyping of these TLR4 mutations and may be useful for evaluating the presence of TLR4 polymorphisms in patients and to predict susceptibility to bacterial infection. Genotyping of 96 samples in a short time period using DNA from various sources is possible with this method. Furthermore, it has a wide dynamic range and therefore is applicable for DNA sets with varying DNA yields and quality.

DNA was extracted from whole blood (n = 120) by use of the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s guidelines. DNA from serum (n = 377) was extracted by use of the Magna Pure LC DNA Isolation Kit I (Roche).

Mutation detection for the LightTyper was based on a reported protocol (. . . [Full Text of this Article]






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