The sensitivity of denaturing gradient gel electrophoresis: a blinded analysis.
Denaturing gradient gel electrophoresis (DGGE) is considered one of the most sensitive and specific of the mutational scanning techniques, yet blinded analyses have not been reported. We report the results of a blinded study of the efficiency of DGGE to detect mutations in the Human Coagulation Factor IX. Two overlapping genomic DNA sequences from exon 8 of Factor IX (290 bp and 539 bp length) with an unknown number of mutations were amplified with a 40 bp GC-clamp and tested blindly by DGGE. DGGE detected all mutations in the 290 bp genomic DNA segment. DGGE detected all but one mutation in the 539 bp genomic segment after experimental conditions were fully optimized but missed multiple mutations in an initial blinded experiment. These results demonstrate the utility of blinded analyses and confirm the exquisite power of DGGE for detecting mutations.
Unit on Molecular Clinical Investigation, NIMH, Bethesda, MD 20892-1384, USA. pgej@helix.nih.gov
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D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons / Q000235:genetics; D005164:Factor IX / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011446:Prospective Studies; D012680:Sensitivity and Specificity; D016037:Single-Blind Method
D005164:Factor IX; D004247:DNA