Allelic discrimination
A fascinating application of the real-time TaqMan PCR is the detection of gene mutations and genome instabilities. The detection of gene mutations is based on the design of two TaqMan probes, specific for the wildtype allele (A) and the mutant allele (B) [39]. Both probes are labelled with two different fluorescent tags. The TaqMan probe is designed with the gene mutation affecting the middle part of the probe sequence. Binding of the wildtype TaqMan probe to the mutant allele and vice versa is suboptimal because of the mismatch within the TaqMan probe and the target sequence. As a consequence, binding of the TaqMan probe to the unmatched target sequence is highly reduced, or even completely abrogated. Reduction of the binding capacity decreases the difference of the reporter fluorescence in the sample and that in the no template control, which is defined as the DRn value. Running appropriate controls for AA, AB and BB, the SDS algorythm for allelic discrimination generates three clusters, and unknown sample DNA will be clearly identified as AA, AB, or BB. A protocol for analysing bovine leucocyte adhesion deficiency (BLAD) has been established [23] and found the system to be highly reliable (observations made by the author; unpublished).

Genome instability includes amplification or deletion of part of the genome resulting in dysmorphology, trisomy or monosomy for parts of the genome. In tumors, the amplification of oncogenes, for example HER2/neu59, or the deletion of tumor suppressor genes, for example p53 [46], are frequently found. Currently, detection of genome instability is accomplished via restriction fragment length polymorphisms (RFLP) or by the different size of PCR products for analysis of a loss in heterozygosity. More laborious methods allow the detection of deletions and amplifications and rely on either quantitative Southern blotting or fluorescent in situ hybridization (FISH). All these methods are difficult to perform, are time-consuming, use large amounts of material or specialised tissue samples and may offer limited resolution.

These difficulties point towards the use of quantitative PCR. Two breast tumor cell lines, MCF-7 and T-47D, were analysed using this method. The T-47D cell line has only one defective copy of p53 [46] and the MCF-7 cell line has only one copy of erbB-211. Both the p53 and the erbB-2 are located on chromosome 17; the S100ß marker located on chromosome 21 was used as a reference. Assays for p53 and S100ß were run in parallel reactions on the same genomic DNA (gDNA) and compared to a control gDNA. By using the ratio between the target and the reference marker, relative ratios of copy numbers can be calculated. Using this technique, gene deletions that had been detected previously, using other techniques, could be successfully detected [12].

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