A Fluorescent Quantitative Multiplex PCR Method to Detect Copy Number Changes in the RB1 Gene

Copy number changes comprising deletions or insertions involving single or multiple exons of a gene are known to occur in a significant proportion of cases in retinoblastoma. The protocol described here involves a two-step quantitative multiplex PCR process which is suitable for the detection of such mutations in the RB1 as well as in other genes. This is achieved through the use of suitable gene-specific primers designed to amplify individual exons, with universal tags attached to the 5' end of each primer. These tagged primers are used in the first step of PCR of the RB1 gene in patients. The second step is carried out through the use of "universal" primers complementary to the tag sequences alone. This technique facilitates the detection of fluorescent PCR products from multiple exons through the use of a single fluorescent tagged primer.