title: Real-time quantitative PCR as an alternative to Southern blot or fluorescence in situ hybridization for detection of gene copy number changes.
abstract: Changes in copy number of genes contribute to the pathogenesis of various genetic disorders and cancer. The status of a gene has not only diagnostic value but sometimes directs treatment stratification. Although, for many years, Southern blot and fluorescence in situ hybridization were the standard methods for the detection of deletion, duplication, or amplification of a gene, both methods have their own important limitations. Recently, realtime quantitative PCR has proven to be a good alternative for the detection of gene copy number changes. Its main advantages are the large dynamic range of accurate quantification, the absence of post-PCR manipulations, its high-throughput screening capacity and degree of automation, and the possibility to perform the assay on minimal amounts of sample DNA in just a few hours of time. In this chapter, we outline the procedure of how to develop an assay for the detection of gene copy number changes for your gene of interest. We illustrate the approach by describing a validated assay for the detection of germline VHL exon deletions and for determination of MYCN copy numbers in tumor samples.
authors.affiliation: Center for Medical Genetics, Ghent University Hospital, Belgium.
authors.forename: Jasmien
authors.initials: J
authors.lastname: Hoebeeck
authors.forename: Frank
authors.initials: F
authors.lastname: Speleman
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
mesh_terms: D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D005091:Exons; D018628:Gene Dosage; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D009690:Nucleic Acid Conformation; D015513:Oncogene Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D017384:Sequence Deletion; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics; D006623:von Hippel-Lindau Disease / Q000235:genetics
chemical_list: D017931:DNA Primers; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D004247:DNA; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human