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Letters |
C) Genotyping
Departments of
1
Clinical Chemistry,
2
Anesthesiology, and
3
Neurology, University Hospital Mannheim of the University of Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany
aAuthor for correspondence. Fax 49-621-383-3819; e-mail thomas.bertsch{at}ikc.ma.uni-heidelberg.de.
To the Editor:
Interleukin-6 (IL-6) is a central protein in the regulation of the inflammatory and immunologic response (1). A G
C polymorphism at position -174 has been associated with osteoporosis (2), juvenile rheumatoid arthritis (3), and atherosclerosis (4) with increased risks in the absence of the CC genotype.
Restriction fragment length polymorphism analysis has been used for IL-6 genotyping, but it is time-consuming and requires multiple manual steps. To improve throughput, we developed a rapid-cycle PCR method on the LightCyclerTM instrument (Roche Diagnostics) with fluorescent probe melting analysis. This assay is completed in 60 min.
DNA from 115 German Caucasians was extracted from whole blood according to standard procedures. Our study population consisted of 40 healthy blood donors and 75 patients from the intensive care unit of our hospital. The reliability of the proposed assay was confirmed by restriction enzyme digestion with SfaNI. PCR was performed in disposable capillaries (Roche Diagnostics) with a reaction volume of 20 µL containing 2 µL of DNA (20–80 ng), 0.5 µM each of the primers (sense, 5'-TTA CTC TTT GTC AAG ACA TGC CA-3'; anti-sense, 5'-ATG AGC CTC AGA CAT CTC CAG-3'), 2 µL of reaction buffer [LightCycler fast start DNA master hybridization probes 10x buffer (1x = 1.75 mM); Roche Diagnostics], 1 µL of MgCl2 (final concentration, 2.25 mM), and 0.2 µM each of the labeled probes. The anchor probe (5'-CTA AGC TGC ACT TTT CCC CCT AGT-3') was labeled at the 3' end with fluorescein. The sensor probe, which is specific for the G allele (5'-GTG TCT TGC GAT GCT AAA GGA-3'), was labeled with LightCycler Red 640 at the 5' end and modified at the 3' end by phosphorylation to block extension. The PCR conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 45 cycles of denaturation (95 °C for 10 s, 20 °C/s), annealing (53 °C for 10 s), and extension (72 °C for 10 s). The melting curve consisted of one cycle at 95 °C for 0 s and 45 °C for 90 s, and then increasing the temperature to 85 °C at a rate of 0.1 °C/s.
The fluorescence signal (F2) was monitored continuously during the temperature ramp and then plotted against the temperature (T). These curves were transformed to derivative melting curves [-d(F2)/dT vs T].
Representative results for the three different genotypes (GG, GC, and CC) are shown in Fig. 1
. Of the 115 samples tested, 33% were GG, 46% were GC, and 21% were CC. In a larger study (383 Caucasians), the most frequent genotype was GC, followed by GG and CC (3). In a direct method comparison, our proposed new technique and the restriction enzyme technique with SfaNI gave identical genotyping results (data not shown). We conclude that the assay is rapid and accurate and seems especially suited for laboratories that process large numbers of samples.
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Acknowledgments
We thank Andreas Nitsche and Olfert Landt (TIB MOLBIOL, Berlin, Germany) for designing the hybridization probes and reading the manuscript.
References
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