Single Strand Conformation Polymorphism

The principle of single-strand conformation polymorphism (SSCP) is the differential gel separation of ssDNA that folds into a specific secondary structure based on its sequence.43-45 For SSCP, the region of interest is amplified and the resulting amplicons are denatured using heat or a denaturation buffer, or both, prior to gel or capillary electrophoresis. Amplicons with different sequences will assume different folding conformations upon dena-turation. Conformational differences reflecting sequence changes are detected as differences in electrophoretic mobility of the ssDNA in a nondenaturing polyacrylamide matrix. In general, a wild-type sample generates two bands, one for each of the two strands of the dsDNA product. Bands of mutant ssDNA migrate to positions different from those of the wild-type ssDNA. A homozygous mutant sample generates two bands, but with different migration patterns from the two wild-type bands. If a heterozygous mutant is present, four bands are generated: two with wildtype mobility and two with mutant mobility. Three also can be observed bands in heterozygous specimens if the mutation changes the conformation of only one strand but not the other. The bands with altered mobility can be isolated from wild-type bands in the gel, allowing even rare somatic mutations in tumors to be sequenced.

Temperature, ionic environment, and pH affect the conformation and therefore must be held constant throughout the SSCP run. Accurate temperature control during SSCP increases reliability and is an easily modifiable parameter in repeatable, nonisotopic experiments that may increase sensitivity. SSCP is adversely affected if unincorporated primers are allowed to bind to the ssDNA during denaturing and cooling prior to electrophoresis, or if nonspecific bands are produced by low-fidelity PCR. In SSCP, elec-trophoretic mobility patterns of variant alleles can be difficult to distinguish from wild type. Another disadvantage is that multiple experimental conditions are required for 100% sensitivity for detection of all sequence variants.

SSCP is most sensitive when the DNA amplicon is less than 200 bp in length. Sensitivity decreases as fragment length increases. This can be overcome by multiplexing differently sized fragments onto a single gel lane and by restriction enzyme digestion prior to electrophoresis. When restriction endonucleases are used, the procedure is referred to as restriction endonuclease fingerprinting-single-strand conformation polymorphism (REF-SSCP). Additionally, SSCP is relatively less sensitive for detecting G to C mutations. The addition of glycerol enhances mutation detection in this circumstance.

Variations of SSCP include RNA-SSCP (rSSCP), dideoxy fingerprinting (ddF), bidirectional ddF (bi-ddF), and SSCP detection of virtually all mutations (DOVAM-SSCP). RNA is more stable and adopts more conformational structures than does ssDNA, allowing enhanced detection using rSSCP. RNA-SSCP is not widely used because of the relative difficulty in producing RNA for analysis. Dideoxy fingerprinting involves a dideoxy Sanger single-primer termination reaction (cycle-sequencing reaction; for additional information on the Sanger reaction, see the section on sequencing, above) followed by nondenaturing elec-trophoresis. A fingerprint bandshift is indicative of sequence changes. Bidirectional ddF is an advancement of ddF whereby the dideoxy Sanger termination reaction is performed with two opposing primers in the same tube. SSCP detection of virtually all mutations is a recently described modification in which SSCP is performed under different conditions with different buffers and gel matrices that result in overall increased sensitivity for mutation identification.

The detection of an altered SSCP pattern does not identify the exact sequence variation present in the analyzed DNA. Therefore, positive SSCP results require DNA sequence analysis to confirm and identify sequence variation.

Examples of Applications of SSCP

1. Screening for mutations in the adenomatous polyposis coli (APC) gene

2. Mutation analysis of the ATP7B gene in Wilson disease

3. Mutation analysis in BRCA1 (in familial breast cancer)

4. Pathogen identification46

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