Biological day-to-day variation and reference change limits of serum cortisol and aldosterone in healthy young men on unrestricted diets.

The positions and distances of melting peaks were identical in individuals with the same genotypes. The detection probe of the apoB3500 mutation was derived from the nonsense DNA strand and matched the wild-type sequence of the gene. Because of the length and base composition of the probe, the melting points were higher compared with the apoE probes. Therefore, the fluorescence signal intensity during PCR reaction was equally high for the DNA sample with the wild-type sequence (curve 3 in Fig. 1, bottom left) and the heterozy-gous individual (curve 2). The melting peaks were clearly differentiated (T m 68 and 71.5 °C). The rather similar melting behavior (compared with apoE) is attributable to the length and base composition of the probes. Using the same procedure as for apoE genotyping, we performed apoB3500 mutation analysis on different wild-type samples (different individuals and different DNA preparations) and samples from a heterozygous individual. The results were identical to the results obtained with gel-based technologies. There was no run-to-run variation of the melting points. Compared with traditional PCR genotyping and mutation detection, applications can be adapted very easily on the LightCycler. We have established additional mutation detection protocols for methylenetetrahydrofolate reduc-tase and prothrombin (manuscript in preparation). In our experience, the basis for the design of the PCR primers is the same as required for traditional thermal cyclers and protocols. Extensive optimization is not required; in fact, for apoB3500, methylenetetrahydrofolate reductase, and prothrombin, the PCR primers and cycle conditions did not need to be optimized at all. Because the DNA sequence of the apoE gene is GC-rich, PCR conditions and primers had to be optimized. Because of the nature of the generation of the fluorescence signal, contaminating, non-specific PCR products do not affect the results. The PCR primers for apoE described in this study generated a specific amplicon. With respect to the hybridization probes, a distance of two nucleotides between the anchor primer and the detection primer was ideal for efficient energy transfer and signal generation. The anchor primer (28 –30 nucleotides in length) should enable hybridization at higher temperatures compared with the detection primer (ϳ20 nucleotides in length). The mutant nucleotide should be positioned close to the middle of the detection primer. It is also important that the annealing temperature in the PCR reaction is kept below the melting point of the detection primer to allow monitoring of the fluorescence during the cycling. In our …