CRITICAL FACTORS IN THE PERFORMANCE AND COST OF 2-DIMENSIONAL GENE SCANNING - RB1 AS A MODEL

Two-dimensional (2-D) gene scanning (TDGS) is a method for mutation de tection based on the electrophoretic separation of PCR-amplified DNA f ragments according to size and base pair sequence. The use of denaturi ng gradient gel electrophoresis (DGGE) as the second separation step p rovides virtually 100% sensitivity, while the 2-D format allows the in spection of multiple gene fragments simultaneously. Analysis of many e xons in parallel is greatly facilitated by extensive PCR multiplexing based on preamplification by long-distance PCR. Recently, TDGS has bee n applied to detect mutations in the retinoblastoma tumor suppressor g ene RB1. Using RB1 as a model, we have now analyzed each step of the p rotocol, presenting overall improvements and a detailed cost analysis, where the total cost of the assay is found to be about $40 (US). An o verall picture of TDGS cost-performance, as compared to direct sequenc ing, is provided as a function of the number of target fragments.