| HOME >> BIOLOGY >> TECHNOLOGY |
Paul Zoller and Theresa Redila-Flores
Bio-Rad Laboratories, Inc., Hercules, CA 94547 USA
Introduction
Denaturing gradient gel electrophoresis (DGGE) is one of several methods
that can be used to screen DNA fragments for small sequence changes or
point mutations. In DGGE, the denaturing environment is created by a combination
of uniform temperature and a linear denaturant gradient formed with urea
and formamide (Fischer and Lerman 1983). A 100% denaturant solution consists
of 7 M urea and 40% formamide. The denaturing gradient may be formed perpendicular
or parallel to the direction of electrophoresis. When running a denaturing
gradient gel, both the amplified mutant and wildtype DNA fragments are
run on the same gel. This way, one can detect a mutation by seeing a band
shift between the mutant and wild-type samples.
The separation principle of DGGE is based on the melting behavior of DNA molecules. In a denaturing gradient acrylamide gel, double-stranded DNA is subjected to a denaturant environment and will melt in discrete segments called melting domains. The melting temperature (Tm) of these domains is sequence-specific. When the Tm of the lowest domain is reached, the DNA will become partially melted, creating branched molecules. Partial melting reduces the DNAs mobility in a polyacrylamide gel. Since the Tm of a particular melting domain is sequence-specific, the presence of a mutation will alter the melting profile when compared to the wild-type DNA. Thus, the DNA containing the mutation will have a different mobility compared to the wild type.
In this experiment, we show that parallel DGGE analysis on
'"/>
Source:
Related biology technology :
1. A Positive Selection Assay for Mutation Analysis in Big Blue Animals
2. Epitope-Tagging Vectors for Functional Analysis in Yeast
3. Map and Link Human Genetic Disorders with SSLP Analysis
4. CastAway Precast Gels for Rapid Automated DNA Sequence Analysis
5. Single-cell RT-PCR Analysis of Paramecium Primaurelia with the Eppendorf cMaster RTplusPCR System
6. Structural Analysis of Glycosylated Peptides in Complex Mixtures with Ion Trap MSn
7. Use of Transcriptor Reverse Transcriptase in Microarray Analysis
8. Increased Sensitivity in Microarray Analysis
9. Gene-Expression Analysis of TP, DPD, and TS Using the LightCycler mRNA Quantification KitsPLUS
10. Agilent 2100 Bioanalyzer Automated Analysis System
11. Analysis of Microarray Data
| Copyright © 2003-2012 Bio-Medicine. All rights reserved. ABOUT | CONTACT US | DISCLAIMER | PRIVACY POLICY | TERMS AND CONDITIONS |  |