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Technical Briefs |
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{at}uni-trier.de
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 (7), but we used SimpleProbe oligomers (Table 1
) instead of pairs of hybridization probes. These singular probes use a terminal self-quenching fluorophore. The quenching moiety changes its properties when stacked with the neighboring base during binding, lowering quenching and increasing the fluorescence signal (515 nm).
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The 28-mer SimpleProbe 299 (A896G), which was identical to the published LightCycler probe (7), failed to yield clear melting data, possibly because of the missing stabilization through the neighboring anchor probe. A longer probe would have bound tighter, but the difference in the melting points of the wild type and variant would have been smaller. To maintain a short sequence and to enhance the binding strength, we introduced some tighter-binding LNA (monomers from Exiqon) bases (9). Substituting 3 or 4 thymidine residues with their LNA analogs and shortening the sequence enabled genotyping of the samples (Table 1
). LNA is a novel nucleic acid analog containing a 2'-4'–bridged ribose that keeps the sugar in an RNA-like structure. DNA-LNA hybrids are reported to be stabilized by 1–3 °C per LNA base pair (Exiqon). The probe sequence contained 65% weak bases; 8 of 20 (40%) were deoxythymidine (dT). The LNA amidite couples slowly, and it is more convenient to include only 1 LNA base on the DNA synthesizer [Expedite; Perseptive (discontinued)]. Substituting the dT base thus gave us the broadest variability to study the influence of an increasing number of incorporated LNA bases. We prepared a few LNA-substituted 20-mer detection probes with identical composition and varied the number of dT-LNA bases from 1 to 4. Melting temperatures (Tms) were determined with a LightCycler dye-labeled anchor probe and long synthetic oligonucleotide targets for both genotypes. The 28-mer reference probe had melting points of 62.2 and 57.4 °C (
Tm = 4.8 °C); the 20-mer probe with 1 dT-LNA substitution melted at 58.1 and 50.6 °C (Tm = 7.5 °C), with 3 substitutions at 61.9 and 54.9 °C (Tm = 7.0 °C), and with 4 substitutions at 64.5 and 57.4 °C (Tm = 7.1 °C). The probe with the single LNA base gave unbalanced peaks and a very low melting peak for the mismatch.
DNA (1 µL of whole blood or 4 µL of serum = 20–40 ng DNA) in a total volume of 10 µL was amplified by use of the LightTyper 96 PCR Kit (Roche) according to the manufacturer’s instructions. The PCR mixture contained PCR buffer [50 mM Tris-HCl, 10 mM KCl, 5 mM (NH4)2SO4, 2 mM MgCl2], nucleotides (0.2 mM each of dATP, dCTP, and dGTP and 0.6 mM dUTP), FastStart Taq DNA polymerase as provided by the manufacturer, 0.5 µM each of forward primer TLR4 S and reverse primer TLR4 A, and 0.2 µM SimpleProbe oligomer Sensor 299 [A]4L, specific for A896G (TIB MOLBIOL; Table 1
). The samples were loaded into a 96-well plate, covered with 10 µL of mineral oil each, and amplified in a GeneAmp PCR System 9600 thermal cycler (Applied Biosystems).
The PCR cycling consisted of a denaturation step at 95 °C for 10 min followed by 50 cycles at 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s. PCR was finished by 72 °C for 4 min. PCR products were cooled to 4 °C, centrifuged (100g for 1 min), and transferred directly to the LightTyper instrument for melting point analysis.
Analysis of the position of C1196T was performed as described above, but using primers TLR4 S and TLR4 R (Table 1
) and a higher annealing temperature of 58 °C. We compared 3 different probes, a 26-mer probe identical to the published sensor LightCycler probe (10), a truncated 19-mer probe with 5'-terminal or 3'-terminal FLQ fluorophore (Table 1
), and a 14-mer probe (not shown). The 19-mer probe with the 3'-terminal fluorophore provided the best results.
Tm analysis was carried out with the LightTyper instrument. Probes were selected to be complementary to the wild type; the free probe is quenched, whereas the bound probe is fluorescent. The probe is released during heating, causing a decrease in fluorescence. The mismatched hybrid is less stable and has a lower Tm. The temperature was increased by 0.1 °C/s from 35 to 70 °C to detect C1196T and from 40 to 70 °C to detect A896G. We applied the assay to DNA extracted from whole blood samples (n = 120) with known genotypes for validation of the new assay and from serum samples (n = 377) with unknown genotypes. The overall detection rate was 100%, and repeated analysis of 10% of the samples confirmed the results for both assays. In the first subset (n = 120), the mean (SD) Tms were 58.19 (0.31) and 51.53 (0.19) °C for the variants of A896G, with a mean (SD) Tm of 6.73 (0.1) °C (Fig. 1
). The LightTyper software determined the genotypes of all samples correctly. There were no negative results or unknown declarations, and only a single analysis was necessary. The results are in accordance with our previous results (7). Our set consisted of 90% homozygous variant A, 10% heterozygous, and no homozygous variant G, confirming this genotype as rare (5)(11).
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The samples containing DNA extracted from serum varied in quality and DNA amounts. The mean (SD) Tms for A and G were 59.35 (0.51) and 52.67 (0.55) °C, respectively, with a mean Tm of 6.77 (0.14) °C. Evaluation of the melting curves gave a distribution of 89.66% homozygous variant A, 10.34% heterozygous, and no homozygous variant G. The automated genotype detection software provided with the LightTyper classified 4.4% of samples as unknown genotype, but visual inspection of samples increased the detection rate to 100%. Analysis of a heterogeneous sample set is therefore improved by personal evaluation in some circumstances, whereas analysis of good-quality DNA can be performed solely by the software.
In addition to polymorphism A896G, samples containing DNA from whole blood (n = 120) were examined for a second TLR4 polymorphism, C1196T. Our aim was to look for linkage of these 2 polymorphisms. Genotyping revealed 90.83% homozygous variant C, 9.17% heterozygous, and no homozygous variants for T. The mean (SD) Tm for C was 58.18 (0.45) °C, and for T was 48.31 (0.41) °C [Tm = 10.01 (0.15) °C]. With the exception of the 1 of 120 individuals being homozygous for 1196C and heterozygous for A896G, we confirmed the linkage (99.17%) of these 2 variants for mid-Europeans (12).
Using a total of 497 heterogeneous DNA samples, we demonstrated that the LightTyper instrument is highly suitable for the analysis of 2 polymorphisms in the TLR4 gene; in contrast to the similar setup using the LightCycler, we had no nonanalyzable samples in the subset containing DNA from whole blood (n = 120). In summary, we simplified and improved our previous detection method for variant A896G (7) by several modifications. (a) We replaced the anchor and sensor with a single probe, thereby reducing chances for mistakes. (b) We increased the sample size from 32 to 96 samples by use of the LightTyper instrument. (c) We improved the Tm separation by introducing an LNA base into the SimpleProbe oligomer, allowing for reliable computerized genotype detection. The assay is robust, and results were highly reproducible based on repeated analysis of 10% of the samples. Even for heterogeneous sample sets varying widely in quality and DNA amounts, personal evaluation of the results was needed only in some cases (4.4%). Although 2 separate analyses showed Tm fluctuations of 
1 °C, the system was very accurate, showing almost no variances within one run, and the resulting temperature differences were very stable. Day-to-day-variability and DNA quality were the most likely factors causing Tm variations.
The single-probe format is advantageous because the molecular neighborhood can be neglected. Hybridization is a complex process, however, and probe selection requires some attention. In this context, LNA bases are a helpful new tool to establish robust assays. We highly recommend this method for TLR4 genotyping and for high-throughput analysis using computerized genotype detection in routine clinical settings.
References
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