Comparative sensitivity of alternative single-strand conformation polymorphism (SSCP) methods.
Single-strand conformation polymorphism (SSCP) analysis is a genetic screening technique that allows for the rapid detection of single nucleotide substitutions in fragments of PCR-amplified genomic DNA or cDNA. Several alternative protocols are now being commonly used to resolve differences in electrophoretic mobility of single-stranded DNA fragments. The aim of this study was to directly compare the sensitivity of three popular SSCP methods using a panel of 19 known human mutations/polymorphisms present in genomic DNA samples. Using a single electrophoresis protocol, 95% of the mutations were detected using small-format PhastGel and the PhastSystem. Large-format gels (5% polyacrylamide and Hydrolink-MDE) were tested both with and without the addition of 10% glycerol. The sensitivity for polyacrylamide and Hydrolink-MDE gels without glycerol was 89% and 79%, respectively, and 68% or 63%, respectively, for glycerol-containing gels. However, all mutations were detected with either polyacrylamide or Hydrolink-MDE when both glycerol and non-glycerol gels were examined. We conclude that comparable very high detection efficiency can be achieved using the PhastSystem or by using a combination of two large-gel conditions with either polyacrylamide or Hydrolink-MDE.
Vidal-Puig
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Beth Israel Hospital, Boston, MA 02215.
Moller
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DE
D006801:Humans; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity