Validation of double gradient denaturing gradient gel electrophoresis through multigenic retrospective analysis

Among established techniques for the identification of either known or new mutations, denaturing gradient gel electrophoresis (DGGE) is one of the mos t effective. However, conventional DGGE is affected by major drawbacks that limit its routine application: the different denaturant gradient ranges an d migration times required for different DNA fragments. We developed a modi fied version of DGGE for high-throughput mutational analysis, double gradie nt DGGE (DG-DGGE), by superimposing a porous gradient over the denaturant g radient, which maintains the zone-sharpening effect even during lengthy ana lyses. Because of this innovation, DG-DGGE achieves the double goals of ret aining full effectiveness in the detection of mutations while allowing iden tical run time conditions for all fragments analyzed. Here we use retrospec tive analysis of a large number of well-characterized mutations and polymor phisms, spanning all predicted melting domains and the whole genomic sequen ce of three different genes-the cystic fibrosis transmembrane conductance r egulator (CFTR), the beta-globin, and the p53 genes-to demonstrate that DG- DGGE may be applied to the rapid scanning of any sequence variation.