COMPARATIVE SENSITIVITY OF ALTERNATIVE SINGLE-STRAND CONFORMATION POLYMORPHISM (SSCP) METHODS

Single-strand conformation polymorphism (SSCP) analysis is a genetic s creening technique that allows for the rapid detection of single nucle otide 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 fragmen ts. The aim of this study was to directly compare the sensitivity of t hree popular SSCP methods using a panel of 19 known human mutations/po lymorphisms present in genomic DNA samples. Using a single electrophor esis protocol, 95% of the mutations were detected using small-format P hastGel(R) and the PhastSystem(TM). Large-format gels (5% polyacrylami de and Hydrolink(R)-MDE(TM)) were tested both with and without the add ition of 10% glycerol. The sensitivity for polyacrylamide and Hydrolin k-MDE gels without glycerol was 89% and 79%, respectively, and 68% or 63%, respectively, for glycerol-containing gels. However; all mutation s were detected with either polyacrylamide or Hydrolink MDE when both glycerol and non-glycerol gels,were examined. We conclude that compara ble very high detection efficiency can be achieved using the PhastSyst em or by using a combination of two large-gel conditions with either p olyacrylamide or Hydrolink-MDE.