A comparison of fluorescent SSCP and denaturing HPLC for high throughput mutation scanning.
We examined 67 different mutations in 16 different amplicons in a comparison of mutation detection by fluorescent single strand conformation polymorphism (F-SSCP) and by denaturing HPLC (DHPLC). F-SSCP was used to analyze fluorescent amplicons with internal size standards and automated fragment analysis (GeneScan, PE Applied Biosystems, Foster City, CA). In DHPLC, unlabelled amplicons were analyzed by reverse phase HPLC with fragment detection by absorbance at 260nm. Both methods had high sensitivity (95-100%) and specificity (100%). Overall, F-SSCP with external temperature control was the more sensitive method, but DHPLC was particularly useful for the rapid analysis of novel fragments.
Ellis
L A
LA
Yorkshire Regional DNA Laboratory, St James's University Hospital, Leeds, United Kingdom.
Taylor
C F
CF
Taylor
G R
GR
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons; D005838:Genotype; D006801:Humans; D008025:Ligases; D009154:Mutation; D018807:Polymorphism, Single-Stranded Conformational; D011506:Proteins / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D006623:von Hippel-Lindau Disease / Q000235:genetics*
C505032:CFTR protein, human; D011506:Proteins; D025521:Tumor Suppressor Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D008025:Ligases; C497446:VHL protein, human