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 language | English |
|---|---|
| Pages (from-to) | 35-40 |
| Number of pages | 6 |
| Journal | Clinical Chemistry |
| Volume | 45 |
| Issue number | 1 |
| Publication status | Published - 1999 |
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ASJC Scopus subject areas
- Clinical Biochemistry
Cite this
Validation of double gradient denaturing gradient gel electrophoresis through multigenic retrospective analysis. / Cremonesi, Laura; Carrera, Paola; Fumagalli, Antonella; Lucchiari, Sabrina; Cardillo, Elena; Ferrari, Maurizio; Righetti, Sabina Carla; Zunino, Franco; Righetti, Pier Giorgio; Gelfi, Cecilia.
In: Clinical Chemistry, Vol. 45, No. 1, 1999, p. 35-40.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Validation of double gradient denaturing gradient gel electrophoresis through multigenic retrospective analysis
AU - Cremonesi, Laura
AU - Carrera, Paola
AU - Fumagalli, Antonella
AU - Lucchiari, Sabrina
AU - Cardillo, Elena
AU - Ferrari, Maurizio
AU - Righetti, Sabina Carla
AU - Zunino, Franco
AU - Righetti, Pier Giorgio
AU - Gelfi, Cecilia
PY - 1999
Y1 - 1999
N2 - 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.
AB - 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.
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M3 - Article
C2 - 9895335
AN - SCOPUS:0032953065
VL - 45
SP - 35
EP - 40
JO - Clinical Chemistry
JF - Clinical Chemistry
SN - 0009-9147
IS - 1
ER -