Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 41)

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Fig. 4.21. Temporal temperature gradient gel. Amplified mutant and wild-type alleles of exon 7 from the cystic
fibrosis gene. Separation by TTGE run at 130 V for 5 hours in 1.25x TAE buffer on a 6 M urea/6% acrylamide gel
(37.5:1) using a temperature range of 50–60 °C and a ramp rate of 2 °C/hr. Lane 1, mutant allele (1154 insTC); lane 2,
mutant allele (G330X); lane 3, mutant allele (deltaF311); lane 4, mutant allele (R334W); and lane 5, wild-type allele.
(Samples courtesy of L. Silverman, Division of Molecular Pathology, University of North Carolina School of Medicine)
Calculating the Run Parameters
To determine the temperature range to use with TTGE, a melting profile of the DNA sequence
should be generated using a DNA melting software program, such as Bio-Rad’s MacMelt
software. As in DGGE, the addition of a 30–40 base pair GC clamp should be added to one of
the PCR primers to insure that the region screened is in the lowest melting domain. The
temperature range for the gradient can be calculated from the melting profile graph by first
determining the lowest and highest non-GC clamp melting temperature of the DNA sequence
(See example in Figure 4.22). From the calculated low and high temperatures, the
theoretical melting temperatures can be lowered by adding urea to the gel. A denaturing urea
gel will lower the theoretical melting temperature of DNA by 2°C for every mole of urea.
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In Figure 4.22, the theoretical melting temperature range on the DNA sequence of interest is
approximately 68 to 82 °C. Therefore, the temperature range should be 54–68°C when using
a 7 M urea gel. TTGE gels typically use 6 M of urea, but for sequences that generate melt
profiles that require buffer temperature greater than 70°C, higher concentrations of urea should
be used. Adding 1–2°C to the final temperature may help to improve the resolution of some
mutations. The typical temperature range for TTGE gels are between 40 and 70°C.
Temperature ramp rates of 1–3°C/hr generally give the best resolution between mutant
and wild-type samples. Slower ramp rates are best, but to reduce run times for routine
screening, ramp rates can be increased empirically. The temperature ramp rate can be
determined if the desired run time or temperature range is known. The ramp rate is calculated
by subtracting the final temperature from the initial temperature and dividing by the desired
run time. In Figure 4.22, if the run time is 4 hours, the ramp rate will be 3°C/hr ([68–54°] ÷ 4 hr
= 3.5 °C/hr). A desired run time is calculated by subtracting the final temperature from the
initial temperature and dividing by the desired ramp rate. In the Figure 4.22 example, if the
ramp rate is 2°C/hr, then the run time will be 7 hours ([68– 54°] ÷ 2 °C/hr = 7 hr).
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