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

Laura Cremonesi, Paola Carrera, Antonella Fumagalli, Sabrina Lucchiari, Elena Cardillo, Maurizio Ferrari, Sabina Carla Righetti, Franco Zunino, Pier Giorgio Righetti, Cecilia Gelfi

Research output: Contribution to journalArticlepeer-review

Abstract

Among established techniques for the identification of either known or new mutations, denaturing gradient gel electrophoresis (DGGE) is one of the most effective. However, conventional DGGE is affected by major drawbacks that limit its routine application: the different denaturant gradient ranges and migration times required for different DNA fragments. We developed a modified version of DGGE for high-throughput mutational analysis, double gradient DGGE (DG-DGGE), by superimposing a porous gradient over the denaturant gradient, which maintains the zone-sharpening effect even during lengthy analyses. Because of this innovation, DG-DGGE achieves the double goals of retaining full effectiveness in the detection of mutations while allowing identical run time conditions for all fragments analyzed. Here we use retrospective analysis of a large number of well-characterized mutations and polymorphisms, spanning all predicted melting domains and the whole genomic sequence of three different genes - the cystic fibrosis transmembrane conductance regulator (CFTR), the β-globin, and the p53 genes - to demonstrate that DG-DGGE may be applied to the rapid scanning of any sequence variation.

Original languageEnglish
Pages (from-to)35-40
Number of pages6
JournalClinical Chemistry
Volume45
Issue number1
Publication statusPublished - 1999

ASJC Scopus subject areas

  • Clinical Biochemistry

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