Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 56)

Fig. 5.5. Pouring a heteroduplex analysis gel.
4. Pour or pipette the gel solution into the sandwich until it 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 6
Single-Stranded Conformational Polymorphisms
6.1 Introduction to SSCP
The SSCP technique is based on the fact that single-stranded DNA has a sequence-specific
secondary structure. Sequence differences as small as a single base change can affect this
secondary structure and can be detected by electrophoresis in a nondenaturing polyacrylamide gel.
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Double-stranded mutant and wild-type samples are first denatured into single strands and then
loaded onto the gel. Differences in mobility of the single strands between the control wild-type DNA
and the other samples indicate a mutation. SSCP is a widely used mutation screening method
because of its simplicity. However, since experimental conditions cannot be predicted for a
particular DNA, it is important to optimize gel electrophoresis conditions. The ability to detect
single base changes rests on several factors which optimize band resolution.
1. Fragment size: The estimated efficiency for detecting single base changes is 90–95% for
fragments less than 350 bp, but the efficiency will decrease as the length of fragment
increases.
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2. Gel temperature: Migration differences due to a single mutation are observed at buffer
temperatures between 4–25°C. Optimal temperature must be determined empirically.
3. Gel additives: In some cases, 5–10% glycerol can be added to the gel to improve the mobility
differences in fragments. Since glycerol can reduce the mobility of single-stranded DNA
fragments at low temperatures, it is typically used with gels run near room temperature.
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