Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 48)

Fig. 4.27. Pouring a TTGE gel.
4. Pour or pipette the gel solution into the sandwich until the gel solution covers the wells of
the comb. Straighten the comb to the desired well depth. Add more solution if needed.
5. Allow the gel to polymerize for about 60 minutes. After polymerization, remove the comb
by pulling it straight up slowly and gently.
6. Continue with Section 8 for electrophoresis.
Section 5
Heteroduplex Analysis
5.1 Introduction to Heteroduplex Analysis
Heteroduplex Analysis (HA) is based on conformational differences in double-stranded
DNA caused by the formation of heteroduplex molecules. 6 Heteroduplex molecules have a
mismatch in the double-strand, causing a distortion in its usual conformation and can be
detected on polyacrylamide gels due to slower migration than the corresponding homoduplex
molecules. Heteroduplex molecules with as little as one mismatch can show a difference in
mobility in a gel than homoduplex molecules. Heteroduplexes are generated in the following
ways: during PCR of a heterozygous individual or by adding mutant and wild-type DNA in
the same PCR reaction or by denaturation and renaturation of mutant and wild-type DNA in
a single tube. Both mutant and wild-type samples are run on the same gel and the mobility of
the fragments is compared.
The sensitivity of heteroduplex analysis is 80–90% in small DNA fragments (< 300 bp). 16
The sensitivity of mutation detection can be improved when used in conjunction with SSCP. 17
A polyacrylamide analog has been developed (MDE ™ or DEM ™ ) which enhances the ability to
detect mutations in heteroduplex samples when compared to conventional polyacrylamide
gels.
18 The addition of urea to the gel can create a mildly denaturing condition which can
increase the separation of heteroduplexes and make mutation detection easier. 16
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