The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA.
Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.
Tu
O
O
Molecular Dynamics, Sunnyvale, CA 94086, USA and Amersham International, Amersham, UK.
Knott
T
T
Marsh
M
M
Bechtol
K
K
Harris
D
D
Barker
D
D
Bashkin
J
J
D002232:Carbocyanines / Q000737:chemistry; D003486:Cyanides / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D015394:Molecular Structure
C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D003486:Cyanides; D005456:Fluorescent Dyes; D004247:DNA