Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 17)

Fig. 4.3. An example of wild-type and mutant DNA fragments that were denatured and re-annealed to
generate four fragments: two heteroduplexes and two homoduplexes run on a parallel
denaturing gradient gel. The melting behavior of the heteroduplexes is altered so that they melt at a lower denaturant
concentration than the homoduplexes and can be visualized on a denaturing gradient gel.
Reagent Preparation
The concentration of denaturant to use varies for the sample being analyzed with the
DCode system. Typically a broad denaturing gradient range is used, such as 0–100% or
20–70%. The concentration of acrylamide can also vary, depending on the size of the
fragment analyzed. Both 0% and 100% denaturant should be made as stock solutions. A
100% denaturant is a mixture of 7 M urea and 40% deionized formamide. Reagents for
casting and running a DGGE gel are included in the DCode Electrophoresis Reagent Kit for
DGGE/CDGE, catalog number 170-9032.
For different percent crosslinking, use the equation below to determine the amount of Bis
to add. The example stock solution below is for an acrylamide/bis ratio of 37.5:1.
40% Acrylamide/Bis (37.5:1)
Reagent
Amount
Acrylamide 38.93 g
Bis-acrylamide 1.07 g
dH
2
O to 100.0 ml
Filter through a 0.45 µ filter and store at 4°C.
13
Denature and reanneal
Wild Type DNA Mutant DNA
wt mut wt + mut
Homoduplexes
Heteroduplexes
Homoduplex
DNA
Heteroduplex
DNA
Wild-Type DNA Mutant DNA
wt + mut wt
20%
60%
mut
Heteroduplex
DNA
Homoduplex
DNA
Homoduplexes
Heteroduplexes
Denature and reanneal
Denaturant