Fluorophore-labeled primers improve the sensitivity, versatility, and normalization of denaturing gradient gel electrophoresis.
Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology. We tested the effect of fluorophore-labeled primers on DGGE band migration, sensitivity, and normalization. The fluorophores Cy5 and Cy3 did not visibly alter DGGE fingerprints; however, 6-carboxyfluorescein retarded band migration. Fluorophore modification improved the sensitivity of DGGE fingerprint detection and facilitated normalization of samples from multiple gels by the application of intralane standards.
Neufeld
Josh D
JD
Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver, British Columbia V6T 1Z3, Canada.
Mohn
William W
WW
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D002232:Carbocyanines; D016172:DNA Fingerprinting / Q000379:methods; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes; D012680:Sensitivity and Specificity; D012988:Soil Microbiology
D002232:Carbocyanines; D017931:DNA Primers; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; C089187:cyanine dye 3; C085321:cyanine dye 5