Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 32)

15. Place the tubing and needle into a beaker of water and reverse the cam on the Gradient
Delivery System. This will rinse the tubing and Y-fitting. Remove both syringes from the
syringe holder on the gradient delivery system. Detach the syringe tubing from the Y-fitting.
Run or push water out through the syringe, tubing, and Y-fitting several times to get rid of any
residual gel solution. It is very important that this is done quickly after casting to avoid
premature gel polymerization.
16. After polymerization, remove the comb by pulling it straight up slowly and gently.
17. Continue with Section 8 for electrophoresis.
4.2 Introduction to Constant Denaturing Gel Electrophoresis (CDGE)
Constant Denaturing Gel Electrophoresis is a modification of DGGE. In CDGE, the
denaturant concentration that gives optimal resolution from a parallel or perpendicular DGGE
gel is held constant.
13 The optimal concentration of denaturant to use for a CDGE is
determined from the maximum split between wild-type and mutant DNA, as seen in the
perpendicular or parallel denaturing gel. To calculate the concentration of denaturant for a
CDGE gel, first place a fluorescent ruler along the axis of the denaturant gradient when taking
a photograph. Then, determine the distance along the gradient where the maximum split is
seen between bands. In the example in Figure 4.14, the distance is 5 cm. Divide this
distance by the length of the gel and multiply by the denaturant range. For example, (5–8) x
50% = 31 %. Add this number to the starting denaturant concentration to determine the
optimum concentration to use for CDGE (20% + 31% = 51%). The same calculation can be
applied to samples that are run on a parallel DGGE gel.
After a mutation has been identified by previous DGGE gels, a CDGE gel can be used to
rapidly screen samples for the presence of a mutation. With no gradient required, rapid, high-
throughput screening is possible. As in DGGE, the formation of heteroduplex analysis can
help in resolving wild-type and mutated fragments when it is not possible to detect a mutation
by running homoduplex fragments. An example of a CDGE gel is shown in Figure 4.15.
Fig. 4.14. Example of perpendicular DGGE gel used for determining the optimum denaturant
concentration used in a CDGE gel. The distance along the gradient where the maximum split seen between
samples is 5 cm. The denaturant concentration of the gradient at this distance is 51%. Therefore, the CDGE gel should
use a denaturant concentration of 51%.
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20% 70%