Serum iron and iron-binding capacity: a round-robin interlaboratory comparison study.

target. Whereas our method involves the additional cost associated with the need for dual-labeling of the probes, it has the advantage of being compatible with performance under standard PCR conditions, regardless of the SNP to be assayed. This characteristic makes it feasible to assay simultaneously, on the same 96or 384-well plate, a variety of SNPs. Interestingly, we noted that our factor V Leiden probe was positioned next to three Gs on the complementary strand. This would mean that the reporter molecule should be partially quenched by those three Gs when the probe anneals to its target (3 ). It is likely, however, that the quenching effect is not sufficient to prevent all fluorescence emission of the reporter because, as Crockett and Wittwer have reported (3 ), this effect has a maximum efficacy of 40%. Finally, although we have noted that the integrity of the probe and the primer ratio imbalance are not absolute prerequisites for the success of the method in individual applications, we have devised a generic protocol that is widely applicable. In addition to factor V Leiden and prothrombin 20210A mutations, for which the results were superimposable with those obtained with the current PCR-restriction fragment length polymorphism method, we have successfully applied it to the detection of a series of other single-base substitutions (data not shown). In summary, we have developed an inexpensive method that combines the use of a single TaqMan probe and melting curves and can be performed under universal conditions.