Double-Gradient DGGE for Optimized Detection of DNA Point Mutations

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Abstract
A novel technique is reported for screening point mutations in genomic DNA: double gradient, denaturing gradient gel electrophoresis (DG-DGGE). Unlike conventional DGGE, which exploits a single gradient of denaturing chemicals (typically urea and formamide) along the migration path to force the two hetero- and two homoduplexes to partially unwind and separate, DG-DGGE superimposes a second (porous) gradient over the denaturing one. With the help of the sieving gradient, molecules such as the hetero-duplexes, which often produce curtains and smears instead of sharp zones, due to lack of a sharp melting transition, are re-compacted into remarkably narrow bands. Even homo-duplexes with minute melting temperature differences, giving a single band in DGGE, are resolved into two zones in DG-DGGE. The technique has been applied to the analysis of a number of point mutations in several exons of the cystic fibrosis transmembrane conductance regulator gene.

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