Quantification of gene modification

The detection and quantification of gene rearrangement, amplification, translocation or deletion is a significant challenge, both in research and in a clinical diagnostic setting. Real-time PCR has become a well-established procedure for quantifying these gene modifications. For example, the major and minor break-points of the chromosome 14 to 18 translocation t(14:18)(q32;q21) have been analyzed in non-Hodgkin's lymphoma using a 5' nuclease assay with TaqMan® probes (Estalilla et al., 2000). The diagnosis and treatment of a number of severe conditions, such as the multi-drug resistance phenotype in tumors, could benefit from the development of a quick assay. Compared to Southern blotting or fluorescence in-situ hybridization (FISH) used for the detection of gene amplification, a SYBR® Green real-time PCR assay provides a rapid and accurate alternative. Furthermore, a FISH-based assay would require biologic material, which may not be available or easy to obtain. Genetic counseling could also benefit from a quick and reliable assay to determine carriers of tumor suppressor gene deletions involved in inherited susceptibility to cancer (e.g. P53, RB, WT1, APC, VHL or BRCA1). The general applicability of a SYBR® Green real-time PCR assay quantifying such genetic events (deletion, amplification and rearrangement) has been demonstrated (Ponchel et al., 2003) and requires inclusion of a number of controls.

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