Advanced topics in BRCA1/BRCA2 analysis by genetic analyzer and real-time PCR

The most common gene changes in breast cancer are those of the BRCA1/BRCA2 genes. As both are large multi-exon genes, mutation screening of BRCA1/BRCA2 is technically challenging, because each gene harbors more than 1,000 different disease-associated mutations, the vast majority of which are individually rare. However, methods widely used in research laboratories such as SSCP, DGCG and HPLC miss nearly one-third of the BRCA mutations that are detected by genetic analyzer. These methods for scanning mutation fail to detect all BRCA1 germline defects. In the meantime, an alternative approach is to use a mutation scanning technique to highlight variations in genomic sequences that are then characterized by genetic analyzer. On the other hand, large genomic rearrangements are not detectable by current PCR-based methods, perhaps explaining the discrepancy between linkage analysis and genetic testing. Quantitative real-time PCR can determine large genomic rearrangements, gene duplications or deletions in BRCA1/BRCA2 genes. Furthermore, melting curve analysis immediately after PCR can identify small mutations, down to single base changes. These techniques are becoming easier and faster and can be multiplexed. In summary, sequencing in combination with real-time PCR methods is a favorable option for the analysis of BRCA genes.