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Temporal Temperature Gradient Electrophoresis: A Powerful Technique to Screen Mutations

Bio-Rad Laboratories, Paul Zoller, Theresa Redila-Flores, Dan Chu, and Asmita Patel, Ph.D.


Introduction
As the number of diseases and disorders linked to gene mutations grow, the need for scanning single-base mutations becomes ever more vital to extending understanding of disease-risk predictor mutations and their outcome. Since most mutations are single base changes, a number of methods that are faster and more economical than DNA sequencing have been developed. Single stranded conformation polymorphism (SSCP),1 denaturing gradient gel electrophoresis (DGGE),2 constant denaturing gel electrophoresis (CDGE),3 temperature gradient gel electrophoresis (TGGE),4 heteroduplex analysis (HA),5 protein truncation test (PTT),6 and enzymatic/chemical cleavage analysis, are the major detection methods used to screen mutations.

The DCode universal mutation detection system (Figure 1) can be used to screen mutations by any of the techniques mentioned above and by a new technique called temporal temperature gradient gel electrophoresis (TTGE).

Temporal temperature gradients previously described by Yoshino et al. and Wiese et al., have been used for electrophoretic separations where the spatial temperature gradient is replaced with a temporal temperature gradient.7,8

In TTGE, the denaturing environment is formed by a constant concentration of denaturant in the gel in combination with the temporal temperature gradient. During electrophoresis, the temperature is increased gradually and uniformly. This results in a linear temperature gradient over the course of the run (Figure 2). Electrophoretic mob
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