Concentration of dye-labeled nucleotides incorporated into DNA determined by surface plasmon resonance-surface plasmon fluorescence spectroscopy.
A method for the accurate determination of the fraction of surface-attached DNA duplexes exhibiting a single-fluorophore-labeled nucleobase is introduced. The fluorescence signals obtained from surface plasmon field-enhanced fluorescence spectroscopy along with the optical properties of the sensor architecture determined by surface plasmon resonance were employed for the calculation. A Cy5-labeled nucleotide was incorporated into DNA at a well-defined position via template-directed DNA synthesis performed by a DNA polymerase. The sample-to-sample variations associated with the optical properties of the employed metal films caused a small variation in the strength of the evanescent field. This variation was accounted for by evanescent field integration over the DNA layers. The exponential-type relationship between the fraction of DNA with Cy5-dCTP incorporation at the surface and the mole fraction of the Cy5-dCTP in solution indicates the preferential incorporation of nonlabeled nucleotides by the DNA polymerase.
Ekgasit
Sanong
S
Sensor Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. sanong.e@ chula.ac.th
Stengel
Gudrun
G
Knoll
Wolfgang
W
D001483:Base Sequence; D015374:Biosensing Techniques; D004396:Coloring Agents; D003596:Cytosine; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000032:analysis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D020349:Surface Plasmon Resonance / Q000379:methods
D004396:Coloring Agents; D009711:Nucleotides; D003596:Cytosine; D004247:DNA