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TTGE on the DCode System

missed. Several of these problems are eliminated by CDGE in which a single denaturing condition is used to melt a fragment.24 In CDGE, wild-type and mutant fragments will melt partially into a certain configuration immediately after entering the gel. This configuration will be kept throughout the run, and the fragments will migrate with a constant rate. Therefore, the longer the gel is run, the wider the separation between the mutant and wild-type samples.


Improvements of DGGE and CDGE by TTGE
CDGE detects nearly all mutations within one melting domain, but has the disadvantage of screening only one domain at a time. Fragments with only one low and one high melting domain will often have a short transition state between a double-stranded and a partially melted state. For example, the theoretical melting curve of a GC-clamped exon 2 sequence from the BRCA1 gene is very flat, containing only one melting domain (Figure 1A). The steep S-shaped curve in the perpendicular DGGE makes it difficult to estimate the optimal denaturing conditions for CDGE (Figure 1B). If the CDGE denaturation conditions are too low or too high, the separation is lost. This is illustrated in Figures 1C and 2C. TTGE is much more robust and solves these problems. In TTGE a single concentration of urea and formamide is used as in CDGE, but the temperature during the run is gradually increased. The denaturant concentration used in TTGE can be determined either from the theoretical melting curve or experimentally from a perpendicular DGGE. A denaturing concentration of approximately 10 units lower than the one corresponding to the steepest part of the curve is used (Figure 1B and 2B). The temperature is
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