Genotypic Approaches

Extensive studies have been undertaken for years to screen the mutations affecting the DPYD gene, and many methods are available now to detect the most common variations in sequence. Most are based upon Real-Time PCR methods (59,83,84,85,86,87) or more complex denaturating HPLC techniques (88,89,90).

The most frequently used methods for detecting SNPs are based upon automated Real-Time-PCR approaches, or alternatively PCR-RFLP (restriction fragment length polymorphism) assays using appropriate restriction enzymes. RT-PCR is based on different melting temperatures of fluorescent-labelled oligonucleotide hybridization probes using a single-step assay combining both fluorescence PCR and melting curve analysis. However, only a priori known mutations can be found using such approaches. Although more complicated and with a longer run time, denaturing-HPLC is capable of detecting all known mutations, but can also identify unknown variations in sequence and promoter hypermethylations (77,90). The rise of high-resolution melting curves generated with new DNA-binding dyes should allow in the near future a total specificity in the screening of mutations with a simple and time-effective RT-PCR-based method.

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